Abeta conformer selective anti-abeta globulomer monoclonal antibodies

ABSTRACT

The subject invention relates to monoclonal antibodies that may be used in the treatment and diagnosis of Alzheimer&#39;s Disease. In particular, the present invention relates to monoclonal antibodies referred to as 10F4 and 3C5 and to other monoclonal antibodies (e.g., murine, human or humanized) having similar properties thereto.

The subject application claims priority to U.S. provisional applicationNo. 60/872,156, filed on Nov. 30, 2006, hereby incorporated herein inits entirety by reference.

BACKGROUND OF THE INVENTION

1. Technical Field

The subject invention relates to monoclonal antibodies that may be usedin the treatment and diagnosis of Alzheimer's Disease. In particular,the present invention relates to monoclonal antibodies referred to as10F4 and 3C5 and to other monoclonal antibodies (e.g., murine, human orhumanized) having similar properties thereto.

2. Background Information

In 1907, the physician Alois Alzheimer first described theneuropathological features of a form of dementia subsequently named inhis honor as Alzheimer's disease (AD). In particular, AD is the mostfrequent cause for dementia among the aged, with an incidence of about100 of the population in those above 65 years of age. With increasingage, the probability of disease also rises. Globally, there are about 15million people affected with the disease and further increases in lifeexpectancy are expected to increase the number of people affected withthe disease to about three-fold over the next decades.

From a molecular point of view, Alzheimer's disease (AD) ischaracterized by a deposit of abnormally aggregated proteins. In thecase of extra-cellular amyloid plaques, these deposits consist mostly ofamyloid-β-peptide filaments, and in the case of the intracellularneurofibrillary tangles (NFTs), mostly of the tau protein. The amyloidβ(Aβ) peptide arises from the β-amyloid precursor protein by proteolyticcleavage. This cleavage is effected by the cooperative activity ofseveral proteases named α-, β- and γ-secretase. Cleavage leads to anumber of specific fragments of differing length. The amyloid plaquesconsist mostly of peptides with a length of 40 or 42 amino acids (Aβ40,Aβ42). The dominant cleavage product is Aβ40; however, Aβ42 has a muchstronger toxic effect. Cerebral amyloid deposits and cognitiveimpairments very similar to those observed in Alzheimer's disease arealso hallmarks of Down's syndrome (trisomy 21), which occurs at afrequency of about 1 in 800 births.

The amyloid cascade hypothesis of Hardy and Higgins postulated thatincreased production of Aβ(1-42) would lead to the formation ofprotofibrils and fibrils (i.e., the principal components of Aβ plaques),these fibrils being responsible for the symptoms of Alzheimer's disease.Despite the poor correlation between severity of dementia and Aβ plaqueburden deposited, this hypothesis was favored until recently. Thediscovery of soluble Aβ forms in AD brains, which correlates better withAD symptoms than plaque load does, has led to a revisedamyloid-cascade-hypothesis.

Active immunization with Aβ peptides leads to a reduction in theformation as well as to partial dissolution of existing plaques. At thesame time, it leads to alleviation of cognitive defects in APPtransgenic mouse models. For passive immunization with antibodiesdirected to Aβ peptides, a reduction of an Aβ plaque burden was alsofound.

The results of a phase IIa trial (ELAN Corporation Plc, South SanFrancisco, Calif., USA and Dublin, UK) of active immunization withAN-1792 (Aβ(1-42) peptide in fibrillary condition of aggregation)suggest that immunotherapy directed to Aβ peptide was successful. In asubgroup of 30 patients, the progression of disease was significantlyreduced in patients with positive anti-Aβ antibody titer, measured byMMSE and DAD index. However, this study was stopped because of seriousside effects in the form of a meningoencephalitis (Bennett and Holtzman,2005, Neurology, 64, 10-12). In particular, meningoencephalitis wascharacterized by neuroinflammation and infiltration of T-cells into thebrain. Presumably, this was due to a T-cell immune response induced byinjection of Aβ(1-42) as antigen. Such an immune response is not to beexpected after passive immunization. To date, there are no clinical datawith reference to this available. However, with reference to such apassive approach to immunization, concerns about the side effect profilewere voiced because of preclinical studies in very old APP23 mice whichreceived an antibody directed against an N-terminal epitope of Aβ(1-42)once a week over 5 months. In particular, these mice showed an increasein the number and severity of microhemorrhages compared to controlanimals treated with saline (Pfeifer et al., 2002, Science, 298, 1379).A comparable increase in microhaemorrhages was also described in veryold (>24 months) Tg2576 and PDAPP mice (Racke et al., 2005, J Neurosci,25, 629-636; Wilcock et al. 2004, J. Neuroinflammation, 1(1):24; DeMattos et al., 2004, Neurobiol. Aging 25(S2):577). In both mousestrains, antibody injection led to a significant increase inmicrohemorrhages. In contrast, an antibody directed against the centralregion of the Aβ(1-42) peptide did not induce microhemorrhages (deMattos et al., supra). The lack of inducing microhemorrhages wasassociated with an antibody treatment which did not bind to aggregatedAβ peptide in the form of CAA (Racke et al., J Neurosci, 25, 629-636).Yet, the exact mechanism leading to microhemorrhages in mice transgenicfor APP has not been understood. Presumably, cerebral amyloid angiopathy(CAA) induces or at least aggravates cerebral hemorrhages. CAA ispresent in nearly every Alzheimer's disease brain and about 20% of thecases are regarded as “severe CAA”. Passive immunization shouldtherefore aim at avoiding microhemorrhages by selecting an antibodywhich recognizes the central or the carboxy terminal region of the Aβpeptide.

International Patent Application Publication No. WO2004/067561 describesstable Aβ(1-42) oligomers (Aβ(1-42) globulomers) and antibodies directedspecifically against the globulomers. Digestion with unspecificproteases shows that the Aβ globulomer may be digested beginning withthe hydrophilic N-terminus protruding from the globular core structure(Barghorn et al., 2005, J Neurochem, 95, 834-847). Such N-terminaltruncated Aβ globulomers (Aβ(12-42) and Aβ(20-42) globulomers) representthe basic structural unit of this oligomeric Aβ and are a very potentantigen for active immunization of rabbits and mice leading to highantibody titers (WO2004/067561). The putative pathological role ofN-terminally truncated Aβ forms in vivo has been suggested by severalrecent reports of their existence in AD brains (Sergeant et al., 2003, JNeurochem, 85, 1581-1591; Thal et al., 1999, J. Neuropathol. Exp Neurol,58, 210-216). During in vivo digestion, certain proteases found inbrain, e.g. neprilysin (NEP 24.11) or insulin degrading enzyme (IDE),may be involved (Selkoe, 2001, Neuron, 32, 177-180).

In view of the above, there is a tremendous and immediate need for atreatment for Alzheimer's Disease which has few, if any, side effects(e.g., microhemmorhages). With such treatment, affected patients may beable to maintain a functional and active lifestyle for many years beyondthat which is possible without such treatment. Thus, not only are therefinancial implications for such a treatment but “quality of life”implications as well, not only for the patients but also for theircaregivers.

All patents and publications referred to herein are hereby incorporatedin their entirety by reference.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1

FIG. 1( a) shows a dot blot analysis of the specificity of differentanti-Aβ antibodies (−6E10, −3C5, 10F4). The monoclonal antibodies testedhere were obtained by active immunization of mice with Aβ(12-42)globulomer (prepared as described in Example I) followed by selection ofthe fused hybridoma cells (except for the commercially available 6E10,Signet, Cat. No.: 9320). The individual Aβ forms were applied in serialdilutions and incubated with the respective monoclonal antibodies forimmune reaction:

-   -   1. Aβ(1-42) monomer, 0.1% NH₄OH    -   2. Aβ(1-40) monomer, 0.1% NH₄OH    -   3. Aβ(1-42) monomer, 0.1% NaOH    -   4. Aβ(1-40) monomer, 0.1% NaOH    -   5. Aβ(1-42) globulomer    -   6. Aβ(12-42) globulomer    -   7. Aβ(20-42) globulomer    -   8. Aβ(1-42) fibril preparation    -   9. sAPPα (Sigma) (first dot: 1 pmol)

FIG. 1( b) illustrates a quantitative evaluation which was done using adensitometric analysis of the intensity. For each Aβ form, only the dotcorresponding to the lowest antigen concentration was evaluated providedthat it had a relative density of greater than 20% of the relativedensity of the last optically unambiguously identified dot of theAβ(1-42) globulomer (threshold). This threshold value was determined forevery dot-blot independently. The value indicates the relationshipbetween recognition of Aβ(1-42) globulomer and the respective Aβ formfor the antibody given.

FIG. 2

FIG. 2( a) represents a detailed description of the patient materialthat was used for analysis.

FIG. 2( b) illustrates the immunoprecipitated amount of Aβ(1-40)-peptideand Aβ(1-42)-peptide as quantified by SELDI-MS analysis for thedifferent patient and control brain samples with the antibodies 6E10,3C5, 10F4 and the control antibody IgG2b.

FIG. 2( c) illustrates the relative immunoprecipitated amount ofAβ(1-40)-peptide and Aβ(1-42)-peptide as quantified by SELDI-MS analysisfor the different patient and control brain samples with the antibodies3C5, 10F4 and the control antibody IgG2b compared to the pan-Aβ-antibody6E10 in percent. The total amount of Aβ-peptide immunoprecipitated byantibody 6E10 was set to 100%.

FIG. 2( d) illustrates the immunoprecipitated amount of Aβ-peptide asquantified by Western blot analysis for the different patient andcontrol brain samples with the antibodies 6E10, 3C5, 10F4 and thecontrol antibody IgG2b.

FIG. 2( e) illustrates the relative immunoprecipitated amount ofAβ-peptide as quantified by Western blot analysis for the differentpatient and control brain samples with the antibodies 3C5, 10F4 and thecontrol antibody IgG2b compared to the pan-Aβ-antibody 6E10 in percent.The total amount of Aβ-peptide immunoprecipitated by antibody 6E10 wasset to 100%.

FIG. 3

FIG. 3( a) shows a Coomassie stained SDS PAGE of:

-   -   1) standard proteins (molecular marker proteins)    -   2) Aβ(1-42) fibril preparation; control    -   3) Aβ(1-42) fibril preparation+mAb 6E10, 20 h, 37° C.,        supernatant    -   4) Aβ(1-42) fibril preparation+mAb 6E10, 20 h, 37° C., pellet    -   5) Aβ(1-42) fibril preparation+mAb 3C5, 20 h, 37° C.,        supernatant    -   6) Aβ(1-42) fibril preparation+mAb 3C5, 20 h 37° C., pellet    -   7) Aβ(1-42) fibril preparation+mAb 10F4, 20 h, 37° C.,        supernatant    -   8) Aβ(1-42) fibril preparation+mAb 10F4, 20 h 37° C., pellet

FIG. 3( b) shows the densitometric quantitative analysis of in vitroantibody binding to Aβ-fibrils.

FIG. 4

FIG. 4( a) represents the verification of amyloid deposits by Congo Redstaining as plaques in brain tissue and as cerebral amyloid angiopathy(CAA) in brain vessels in the APP transgenic mouse line Tg2576 and in anAD patient (RZ55).

FIG. 4( b) shows the strong staining of parenchymal deposits ofAβ(amyloid plaques) in an AD patient (RZ16) occurs only with 6G1 and thecommercially available antibody 6E10 while 10F4 and 3C5 showconsiderably weaker staining. All antibodies were tested at aconcentration of 0.7 μg/mL.

FIG. 4( c) shows the strong staining of parenchymal deposits ofAβ(amyloid plaques) in TG2576 mice occurs only with 6G1 and thecommercially available antibody 6E10 while 10F4 and 3C5 showconsiderably weaker staining. All antibodies were tested at aconcentration of 0.7 μg/mL.

FIGS. 4( d)-4(g) show the quantification of the analysis of Aβ plaquestaining in the histological images using image analysis. Opticaldensity values (0%=no staining) were calculated from the greyscalevalues of plaques subtracted by greyscale values of background tissue.(FIG. 4( d) shows the binding of 0.7 μg/mL antibody in Tg2576 mice. FIG.4( e) shows the binding of 0.07-0.7 μg/mL antibody in APP/L mice. FIG.4( f) shows the binding of 0.7 μg/mL antibody in an AD patient (RZ55),and FIG. 4( g) shows the binding of 0.07-0.7 μg/mL antibody in an ADpatient (RZ16).) The differences between staining of the commerciallyavailable antibodies 6E10 (starts) and 4G8 (circles) and antibodies 6G1,10F4 and 3C5 (one asterisk/circle: p<0.05, two asterisks/circles:p<0.01, and three asterisks/circles: p<0.001 versus control; post-hocBonferroni's t-test after ANOVA with p<0.001) were statisticallyevaluated (FIGS. 4( d) and (e)). In FIGS. 4( e) and 4(g), the antibodies10F4 and 3C5 showed always significantly less staining than thecommercially available antibodies 6E10 and 4G8 (p<0.05 in post-hoct-test after p<0.001 in ANOVA).

FIG. 4( h) shows the strong staining of vascular deposits of Aβ(arrows)occurs only with 6G1 and the commercially available antibody 6E10 whilestaining with 8F5 or 8C5 was much weaker. All antibodies were tested ata concentration of 0.7 μg/mL. A qualitatively similar situation wasfound in Tg2576 mice (not shown here).

FIG. 5

FIGS. 5( a), (c), (e) and (g) show the amount of Aβ(1-40) and Aβ(1-42)peptide immunoprecipitated from Alzheimer's disease patient CSF by themonoclonal antibodies 6E10, 10F4, 3C5 and 8F5. Results for 4 individualAlzheimer's disease CSF samples are shown ((a)=Alzheimer's diseasepatient #0504009; (c)=Alzheimer's disease patient #30027;(e)=Alzheimer's disease patient #30026; (g)=Alzheimer's disease patient#26748015).

FIG. 5( b) shows the relative amount of Aβ(1-40) and Aβ(1-42) peptideimmunoprecipitated from Alzheimer's disease patient CSF by theantibodies 10F4, 3C5 and 8F5 compared to the amount of Aβ-peptideimmunoprecipitated by the antibody 6E10 in percent. The total amount ofAβ-peptide immunoprecipitated by mAb 6E10 antibody was set to 1000.Results for 4 individual Alzheimer's disease CSF samples are shown((b)=Alzheimer's disease patient #0504009; (d)=Alzheimer's diseasepatient #30027; (f)=Alzheimer's disease patient #30026; (h)=Alzheimer'sdisease patient #26748015). FIG. 5( i) represents a detailed descriptionof the Alzheimer's disease patient CSF material that was used foranalysis in FIGS. 5( a)-5(i).

FIG. 6

FIG. 6( a) illustrates the DNA sequence (SEQ ID NO:1) of the variableheavy chain encoding the monoclonal antibody referred to herein as“3C5”.

FIG. 6( b) illustrates the DNA sequence (SEQ ID NO:2) of the variablelight chain encoding the monoclonal antibody referred to herein as“3C5”.

FIG. 6( c) illustrates the DNA sequence (SEQ ID NO:3) of the variableheavy chain encoding the monoclonal antibody referred to herein as“10F4”.

FIG. 6( d) illustrates the DNA sequence (SEQ ID NO:4) of the variablelight chain encoding the monoclonal antibody referred to herein as“10F4”.

FIG. 7

FIG. 7( a) illustrates the amino acid sequence (SEQ ID NO:5) of thevariable heavy chain encoding the monoclonal antibody referred to hereinas “3C5”.

FIG. 7( b) illustrates the amino acid sequence (SEQ ID NO:6) of thevariable light chain encoding the monoclonal antibody referred to hereinas “3C5”.

FIG. 7( c) illustrates the amino acid sequence (SEQ ID NO:7) of thevariable heavy chain encoding the monoclonal antibody referred to hereinas “10F4”.

FIG. 7( d) illustrates the amino acid sequence (SEQ ID NO:8) of thevariable light chain encoding the monoclonal antibody referred to hereinas “10F4”. (Complementarity determining regions (CDRs) are underlined ineach described sequence.)

SUMMARY OF THE INVENTION

The present invention encompasses antibodies, directed against Aβglobulomers, which improve the cognitive performance of a patient inimmunotherapy, while at the same time reacting only with a small portionof the entire amount of Aβ peptide in the brain. Such properties preventa substantial disturbance of cerebral Aβ balance and lead to less sideeffects. (For instance, a therapeutically questionable reduction ofbrain volume has been observed in the study of active immunization withAβ peptides in fibrillary condition of aggregation (ELAN CorporationPlc, South San Francisco, Calif., USA and Dublin, UK) of activeimmunization with AN-1792 (Aβ(1-42) peptide in fibrillary condition ofaggregation). Moreover, in this trial, severe side effects in form of ameningoencephalitis were observed.)

In particular, the present invention solves the above-noted side effectissues by providing Aβ globulomer antibodies possessing high affinityfor Aβ globulomers. These antibodies are capable of discriminating otherforms of Aβ peptides, particularly monomers, fibrils and sAPPα. Further,the antibodies of the present invention also discriminate againstamyloid beta in the cerebrospinal fluid (CSF) by binding only to non-CSFamyloid beta. Additionally, the antibodies of the present invention(e.g., 10F4 and 3C5) bind less to Aβ-plaques and vascular Aβ compared toa known antibody (i.e., 6E10).

In particular, the present invention encompasses an isolated antibodyhaving a higher affinity to Aβ(1-42) globulomer than to at least oneamyloid beta protein selected from the group consisting of Aβ(1-42)peptide present in cerebrospinal fluid (CSF) and b) Aβ(1-40) peptidepresent in CSF.

The present invention also includes an isolated antibody having abinding affinity to Aβ(1-42) globulomer which is greater than thebinding affinity to at least one amyloid beta protein selected from thegroup consisting of a) Aβ(1-42) monomer, b) Aβ(1-40) monomer, c)Aβ(1-42) fibril and d) soluble amyloid precursor protein-alpha (sAPPα).This antibody binds with greater affinity to amyloid beta proteinpresent in non-CSF than to amyloid beta protein present in CSF.

The above-described antibodies may be, for example, murine, monoclonal,recombinant, human and/or humanized. Further, any one of more of theantibodies of the present invention may bind to at least one epitope,which is the same epitope or epitopes, to which the monoclonal antibody10F4 (obtainable from a hybridoma designated by American Type CultureCollection deposit number PTA-7808) or the monoclonal antibody 3C5(obtainable from a hybridoma designated by American Type CultureCollection deposit number PTA-7406) binds.

Additionally, the present invention includes an isolated antibodycomprising SEQ ID NO:5, an isolated antibody comprising SEQ ID NO:6 andan isolated antibody comprising both SEQ ID NO:5 and SEQ ID NO:6.

Further, the present invention encompasses an isolated antibodycomprising SEQ ID NO:7, an isolated antibody comprising SEQ ID NO:8 andan isolated antibody comprising both SEQ ID NO:7 and SEQ ID NO:8.

The above-described antibodies of the present invention may comprise atleast one amino acid sequence selected from the group consisting of: a)the amino acid sequence of the heavy chain CDR3 and the amino acidsequence of the light chain CDR3 of monoclonal antibody (10F4)(obtainable from a hybridoma designated by American Type CultureCollection deposit number PTA-7808) and b) the amino acid sequence ofthe heavy chain CDR3 and the amino acid sequence of the light chain CDR3of monoclonal antibody (3C5) (obtainable from a hybridoma designated byAmerican Type Culture Collection deposit number PTA-7406).

Further, the above-described antibodies of the present invention maycomprise at least one amino acid sequence selected from the groupconsisting of: a) the amino acid sequence of the heavy chain CDR2 andthe amino acid sequence of the light chain CDR2 of a monoclonal antibody(10F4) (obtainable from a hybridoma designated by American Type CultureCollection deposit number PTA-7808) and b) the amino acid sequence ofthe heavy chain CDR2 and the amino acid sequence of the light chain CDR2of a monoclonal antibody (3C5) (obtainable from a hybridoma designatedby American Type Culture Collection deposit number PTA-7406).

Also, the antibodies of the present invention may comprise at least oneamino acid sequence selected from the group consisting of: a) the aminoacid sequence of the heavy chain CDR1 and the amino acid sequence of thelight chain CDR1 of a monoclonal antibody (10F4) (obtainable from ahybridoma designated by American Type Culture Collection deposit numberPTA-7808) and b) the amino acid sequence of the heavy chain CDR1 and theamino acid sequence of the light chain CDR1 of a monoclonal antibody(3C5) (obtainable from a hybridoma designated by American Type CultureCollection deposit number PTA-7406).

Moreover, the present invention also includes an isolated antibodycomprising at least one CDR selected from the group consisting of aminoacid sequence: a) SHYAWN; b) YIDYSGSTRYLPSLKS; c) GSGYFYGMDY; d)HASQNINVWLS; e) KASNLHT; f) QQGQSYPYT; g) NYLIE; h) VINPGSGDTNYNENFKG;i) GVITTGFDY; j) RASGNIHNYLA; k) NAKTLAD and l) QHFWSSPRT.

Additionally, the present invention encompasses a hybridoma designatedby American Type Culture Collection deposit number PTA-7808 as well as amonoclonal antibody (10F4) obtainable from or produced by a hybridomadesignated by American Type Culture Collection deposit number PTA-7808.

The invention also includes a hybridoma designated by American TypeCulture Collection deposit number PTA-7406 as well as a monoclonalantibody (3C5) obtainable from or produced by a hybridoma designated byAmerican Type Culture Collection deposit number PTA-7406.

Furthermore, the present invention includes an isolated nucleic acidmolecule encoding the antibodies described above. The nucleotidesequence of this molecule may comprise at least one sequence selectedfrom the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ ID NO:3 andSEQ ID NO:4. Also, the present invention includes a vector comprisingthe isolated nucleic acid molecule as well as a host cell comprising thevector.

Additionally, the present invention includes a method of producing anantibody, comprising culturing the host cell described above in aculture medium for a time and under conditions suitable for productionof any one of the antibodies described above. The antibody produced inaccordance with this method is also included within the scope of thepresent invention.

Also, the present invention includes a composition comprising any one ormore of the antibodies described above. This composition may furthercomprise a pharmaceutically acceptable carrier.

Further, the present invention encompasses a monoclonal antibodycomprising an amino acid sequence encoded by at least one nucleotidesequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2,SEQ ID NO:3 and SEQ ID NO:4. This antibody may be selected from thegroup consisting of a monoclonal antibody produced by a hybridomadesignated by American Type Culture Collection deposit number PTA-7406and a monoclonal antibody produced by a hybridoma designated by AmericanType Culture Collection deposit number PTA-7808. Also, the antibody maycomprise at least one amino acid sequence selected from the groupconsisting of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8.

The invention also includes a method for treating or preventing anamyloidosis in a patient in need of such treatment or prevention. Thismethod comprises administering one or more of the above-describedantibodies (via passive immunization) to the patient in an amountsufficient to effect treatment or prevention. The amyloidosis may be,for example, Alzheimer's disease or the amyloidosis of Down's syndrome.

Also, the present invention encompasses an isolated antibody which bindsto at least one epitope of amyloid beta protein in the brain of apatient having amyloidosis. This antibody may be produced, for example,by a hybridoma having an ATCC deposit number selected from the groupconsisting of PTA-7406 and PTA-7808.

The present invention also includes a method of diagnosing Alzheimer'sDisease in a patient suspected of having this disease. This methodcomprises the steps of isolating a biological sample (for example, a CSFsample or brain tissue sample) from the patient, contacting thebiological sample with one or more of the antibodies described above fora time and under conditions sufficient for formation of antigen/antibodycomplexes, and detecting presence of the antigen/antibody complexes inthe sample, presence of the complexes indicating a diagnosis ofAlzheimer's Disease in the patient. The antigen of the complex may be,for example, a globulomer.

Additionally, the present invention encompasses another method ofdiagnosing Alzheimer's Disease in a patient suspected of having thisdisease. This method comprises the steps of isolating a biologicalsample from the patient, contacting the biological sample with anantigen for a time and under conditions sufficient for the formation ofantibody/antigen complexes, adding a conjugate to the resultingantibody/antigen complexes for a time and under conditions sufficient toallow the conjugate to bind to the bound antibody (wherein the conjugatecomprises an isolated antibody of the present invention attached to asignal generating compound capable of generating a detectable signal),and detecting the presence of an antibody which may be present in thebiological sample by detecting a signal generated by the signalgenerating compound, the signal indicating a diagnosis of Alzheimer'sDisease in the patient. The antigen used in the assay may be, forexample, a globulomer.

Further, the present invention includes an additional method ofdiagnosing Alzheimer's Disease in a patient suspected of havingAlzheimer's Disease. This method comprises the steps of isolating abiological sample from the patient, contacting the biological samplewith an anti-antibody (wherein the anti-antibody is specific for one ofmore of the antibodies of the present invention), for a time and underconditions sufficient to allow for formation of anti-antibody/antibodycomplexes, the complexes containing antibody present in the biologicalsample, adding a conjugate to the resulting anti-antibody/antibodycomplexes for a time and under conditions sufficient to allow theconjugate to bind to bound antibody (wherein the conjugate comprises anantigen, which binds to a signal generating compound capable ofgenerating a detectable signal), and detecting a signal generated by thesignal generating compound, this signal indicating a diagnosis ofAlzheimer's Disease in the patient.

Additionally, the present invention includes a vaccine comprising one ormore of the antibodies of the present invention and a pharmaceuticallyacceptable adjuvant.

Further, the present invention encompasses a method of identifyingcompounds suitable for active immunization of a patient predicted todevelop Alzheimer's Disease. This method comprises the steps of exposingone or more compounds of interest to one or more of the antibodies ofthe present invention, for a time and under conditions sufficient forthe one or more compounds to bind to the one or more antibodies and thenidentifying those compounds which bind to the one or more antibodies,the identified compounds to be used in active immunization in a patientpredicated to develop Alzheimer's Disease.

Also, the present invention includes a kit comprising one or more of theantibodies of the present invention and a conjugate comprising anantibody attached to a signal-generating compound, wherein the antibodyof the conjugate is different from the one or more antibodies within thekit. A package insert may also be included in the kit which describesthe procedure to be utilized in carrying out the assay as well as thecomponents of the kit.

The present invention also includes another kit comprising ananti-antibody to one or more antibodies of the present invention and aconjugate comprising an antigen attached to a signal-generatingcompound. The antigen may be, for example, a globulomer. Again, apackage insert may be included which describes the steps to be utilizedin carrying out the assay as well as the components of the kit.

DETAILED DESCRIPTION OF THE INVENTION

The antibodies of the present invention were designed from immunizationwith the truncated globulomer Aβ(12-42) as described in Example 1. Inparticular, monoclonal antibodies 3C5 and 10F4 were generated againstthe truncated (12-42)-globulomer (in contrast to monoclonal antibodies8F5 and 8C5 which have been made against the Aβ(1-42) globulomer). ThisAβ(12-42) globulomer was made directly from Aβ 12-42 peptide in contrastto the procedure described in Barghorn et al. (J. Neurochem, 95,834-847) and in Example 3, Section 6, wherein the (12-42) globulomer wasmade from pre-existing 1-42-globulomer by limited proteolysis. These twoAβ(12-42) globulomer variants differ in their final aggregation pattern.The one made from Aβ(12-42) peptide shows only the intermediateglobulomer forms (“oligomer A” as described in WO2004/067561) and theone made from the pre-existing Aβ(1-42)-globulomer is the matureglobulomer (“oligomer B” as described in WO2004/067561).

It is an object of the present invention to provide antibodies directedagainst Aβ globulomers which improve the cognitive performance of apatient in immunotherapy while at the same time reacting only with asmall portion of the entire amount of Aβ peptide in brain. This isexpected to prevent a substantial disturbance of cerebral Aβ balance andlead to less side effects. (For instance, as noted above, atherapeutically questionable reduction of brain volume has been observedin the study of active immunization with Aβ peptides in fibrillarycondition of aggregation (ELAN trial with AN1792). Moreover, in thistrial severe side effects in form of a meningoencephalitis wereobserved. The present invention solves this problem by providingglobulomer-specific antibodies possessing high affinity for Aβglobulomers. These antibodies are capable of discriminating other formsof Aβ peptides, particularly monomers and fibrils. Further, theseantibodies do not bind (or bind with a lower affinity compared tocommercially available antibodies (such as 6E10) (Signet Cat. no.:9320)) to amyloid beta in cerebral spinal fluid. Consequently, thepresent invention relates to an antibody having a binding affinity to Aβglobulomer

The term “Aβ(X-Y)” herein refers to the amino acid sequence from aminoacid position X to amino acid position Y of the human amyloid p proteinincluding both X and Y, in particular to the amino acid sequence fromamino acid position X to amino acid position Y of the amino acidsequence DAEFRHDSGY EVHHQKLVFF AEDVGSNKGA IIGLMVGGVV IAT (correspondingto amino acid positions 1 to 43) or any of its naturally occurringvariants, in particular those with at least one mutation selected fromthe group consisting of A2T, H6R, D7N, A21G (“Flemish”), E22G(“Arctic”), E22Q (“Dutch”), E22K (“Italian”), D23N (“Iowa”), A42T andA42V wherein the numbers are relative to the start of the Aβ peptide,including both position X and position Y or a sequence with up to threeadditional amino acid substitutions, none of which may preventglobulomer formation, preferably with no additional amino acidsubstitutions in the portion from amino acid 12 or X, whichever numberis higher, to amino acid 42 or Y, whichever number is lower, morepreferably with no additional amino acid substitutions in the portionfrom amino acid 20 or X, whichever number is higher, to amino acid 42 orY, whichever number is lower, and most preferably with no additionalamino acid substitutions in the portion from amino acid 20 or X,whichever number is higher, to amino acid 40 or Y, whichever number islower, an “additional” amino acid substation herein being any deviationfrom the canonical sequence that is not found in nature.

The term “Aβ(1-42)” herein refers to the amino acid sequence from aminoacid position 1 to amino acid position 42 of the human amyloid β proteinincluding both 1 and 42, in particular to the amino acid sequenceDAEFRHDSGY EVHHQKLVFF AEDVGSNKGA IIGLMVGGVV IA or any of its naturallyoccurring variants, in particular those with at least one mutationselected from the group consisting of A2T, H6R, D7N, A21G (“Flemish”),E22G (“Arctic”), E22Q (“Dutch”), E22K (“Italian”), D23N (“Iowa”), A42Tand A42V wherein the numbers are relative to the start of the Aβpeptide, including both 1 and 42 or a sequence with up to threeadditional amino acid substitutions none of which may prevent globulomerformation, preferably with no additional amino acid substitutions in theportion from amino acid 20 to amino acid 42. Likewise, the term“Aβ(1-40)” here refers to the amino acid sequence from amino acidposition 1 to amino acid position 40 of the human amyloid β proteinincluding both 1 and 40, in particular to the amino acid sequenceDAEFRHDSGY EVHHQKLVFF AEDVGSNKGA IIGLMVGGVV or any of its naturallyoccurring variants, in particular those with at least one mutationselected from the group consisting of A2T, H6R, D7N, A21G (“Flemish”),E22G (“Arctic”), E22Q (“Dutch”), E22K (“Italian”), and D23N (“Iowa”)wherein the numbers are relative to the start of the Aβ peptide,including both 1 and 40 or a sequence with up to three additional aminoacid substitutions none of which may prevent globulomer formation,preferably with no additional amino acid substitutions in the portionfrom amino acid 20 to amino acid 40.

The term “Aβ(12-42)” here refers to the amino acid sequence from aminoacid position 12 to amino acid position 42 of the human amyloid βprotein including both 12 and 42, in particular to the amino acidsequence VHHQKLVFF AEDVGSNKGA IIGLMVGGVV IA or any of its naturallyoccurring variants, in particular, those with at least one mutationselected from the group consisting of A21G (“Flemish”), E22G (“Arctic”),E22Q (“Dutch”), E22K (“Italian”), D23N (“Iowa”), A42T and A42V whereinthe numbers are relative to the start of the Aβ peptide, including both12 and 42 or a sequence with up to three additional amino acidsubstitutions none of which may prevent globulomer formation, preferablywith no additional amino acid substitutions in the portion from aminoacid 20 to amino acid 42.

The term “Aβ(20-42)” herein refers to the amino acid sequence from aminoacid position 20 to amino acid position 42 of the human amyloid βprotein including both 20 and 42, in particular, to the amino acidsequence F AEDVGSNKGA IIGLMVGGVV IA or any of its naturally occurringvariants, in particular those with at least one mutation selected fromthe group consisting of A21G (“Flemish”), E22G (“Arctic”), E22Q(“Dutch”), E22K (“Italian”), D23N (“Iowa”), A42T and A42V wherein thenumbers are relative to the start of the Aβ peptide, including both 20and 42 or a sequence with up to three additional amino acidsubstitutions none of which may prevent globulomer formation, preferablywithout any additional amino acid substitutions.

The term “Aβ(X-Y) globulomer” (Aβ(X-Y) globular oligomer) herein refersto a soluble, globular, non-covalent association of Aβ(X-Y) peptides asdefined above, possessing homogeneity and distinct physicalcharacteristics. According to one aspect, the Aβ(X-Y) globulomers arestable, non-fibrillar, oligomeric assemblies of Aβ(X-Y) peptides whichare obtainable by incubation with anionic detergents. In contrast tomonomers and fibrils, these globulomers are characterized by definedassembly numbers of subunits (e.g. early assembly forms, n=4-6,“oligomers A”, and late assembly forms, n=12-14, “oligomers B”, asdescribed in WO2004/067561). The globulomers have a 3-dimensionalglobular type structure (“molten globule”, see Barghorn et al., 2005, JNeurochem, 95, 834-847). They may be further characterized by one ormore of the following features:

cleavability of N-terminal amino acids X-23 with promiscuous proteases(such as thermolysin or endoproteinase GluC) yielding truncated forms ofglobulomers;

non-accessibility of C-terminal amino acids 24-Y with promiscuousproteases and antibodies;

truncated forms of these globulomers maintain the 3-dimensional corestructure of said globulomers with a better accessibility of the coreepitope Aβ(20-Y) in its globulomer conformation.

According to the invention and, in particular, for the purpose ofassessing the binding affinities of the antibodies of the presentinvention, the term “Aβ(X-Y) globulomer” herein refers, in particular,to a product which is obtainable by a process as described, for example,in Example I presented below. (See also WO 04/067561.) Such a processmay be used to obtain Aβ(1-42) globulomers, Aβ(12-42) globulomers, andAβ(20-42) globulomers. Preferably, the globulomer shows affinity toneuronal cells. Preferably, the globulomer also exhibits neuromodulatingeffects. According to another aspect of the invention, the globulomerconsists of 11 to 16, and most preferably, of 12 to 14 Aβ(X-Y) peptides.

According to another aspect of the invention, the term Aβ(X-Y)globulomer” herein refers to a globulomer consisting essentially ofAβ(X-Y) subunits, where it is preferred if on average at least 11 of 12subunits are of the Aβ(X-Y) type, more preferred if less than 10% of theglobulomers comprise any non-Aβ(X-Y) peptides, and most preferred if thecontent of non-Aβ(X-Y) peptides is below the detection threshold. Morespecifically, the term “Aβ(1-42) globulomer” herein refers to aglobulomer consisting essentially of Aβ(1-42) units as defined above;the term “Aβ(12-42) globulomer” herein refers to a globulomer consistingessentially of Aβ(12-42) units as defined above; and the term “Aβ(20-42)globulomer” herein refers to a globulomer consisting essentially ofAβ(20-42) units as defined above.

The term “cross-linked Aβ(X-Y) globulomer” as used herein refers to amolecule obtainable from an Aβ(X-Y) globulomer as described above bycross-linking, preferably chemically cross-linking, more preferably,aldehyde cross-linking, most preferably, glutardialdehyde cross-linkingof the constituent units of the globulomer. In another aspect of theinvention, a cross-linked globulomer is essentially a globulomer inwhich the units are at least partially joined by covalent bonds, ratherthan being held together by non-covalent interactions only. For thepurposes of the present invention, a cross-linked Aβ(1-42) globulomeris, in particular, a cross-linked Aβ(1-42) oligomer.

The term “Aβ(X-Y) globulomer derivative” as used herein refers, inparticular, to a globulomer that is labelled by being covalently linkedto a group that facilitates detection, preferably, a fluorophore, e.g.,fluorescein isothiocyanate, phycoerythrin, Aequorea victoria fluorescentprotein, Dictyosoma fluorescent protein or any combination orfluorescence-active derivative thereof; a chromophore; achemoluminophore, e.g., luciferase, preferably Photinus pyralisluciferase, Vibrio fischeri luciferase, or any combination orchemoluminescence-active derivative thereof; an enzymatically activegroup, e.g., peroxidase, e.g., horseradish peroxidase, or anyenzymatically active derivative thereof; an electron-dense group, e.g.,a heavy metal containing group, e.g., a gold containing group; a hapten,e.g., a phenol derived hapten; a strongly antigenic structure, e.g.,peptide sequence predicted to be antigenic, e.g., predicted to beantigenic by the algorithm of Kolaskar and Tongaonkar; an aptamer foranother molecule; a chelating group, e.g., hexahistidinyl; a natural ornature-derived protein structure mediating further specificprotein-protein interactions, e.g., a member of the fos/jun pair; amagnetic group, e.g., a ferromagnetic group; or a radioactive group,e.g., a group comprising ¹H, ¹⁴C, ³²P, ³⁵S or ¹²⁵I or any combinationthereof; or to a globulomer flagged by being covalently or bynon-covalent high-affinity interaction, preferably covalently linked toa group that facilitates inactivation, sequestration, degradation and/orprecipitation, preferably flagged with a group that promotes in vivodegradation, more preferably with ubiquitin, where is particularlypreferred if this flagged oligomer is assembled in vivo; or to aglobulomer modified by any combination of the above. Such labelling andflagging groups and methods for attaching them to proteins are known inthe art. Labelling and/or flagging may be performed before, during orafter globulomerization. In another aspect of the invention, aglobulomer derivative is a molecule obtainable from a globulomer by alabelling and/or flagging reaction. Correspondingly, term “Aβ(X-Y)monomer derivative” herein refers, in particular, to an Aβ monomer thatis labelled or flagged as described for the globulomer.

The term “greater affinity” herein refers to a degree of interactionwhere the equilibrium between unbound antibody and unbound globulomer onthe one hand and antibody-globulomer complex on the other is further infavor of the antibody-globulomer complex. Likewise, the term “smalleraffinity” herein refers to a degree of interaction where the equilibriumbetween unbound antibody and unbound globulomer on the one hand andantibody-globulomer complex on the other is further in favour of theunbound antibody and unbound globulomer. The term “greater affinity” issynonymous with the term “higher affinity” and term “smaller affinity”is synonymous with the term “lower affinity”.

The term “Aβ(X-Y) monomer” herein refers to the isolated form of theAβ(X-Y) peptide, preferably, a form of the Aβ(X-Y) peptide which is notengaged in essentially non-covalent interactions with other Aβ peptides.Practically, the Aβ(X-Y) monomer is usually provided in the form of anaqueous solution. In a particularly preferred embodiment of theinvention, the aqueous monomer solution contains 0.05% to 0.2%, morepreferably, about 0.1% NH₄OH. In another particularly preferredembodiment of the invention, the aqueous monomer solution contains 0.05%to 0.2%, more preferably, about 0.1% NaOH. When used (for instance, fordetermining the binding affinities of the antibodies of the presentinvention), it may be expedient to dilute said solution in anappropriate manner. Further, it is usually expedient to use saidsolution within 2 hours, in particular, within 1 hour, and especiallywithin 30 minutes after its preparation.

The term “fibril” herein refers to a molecular structure that comprisesassemblies of non-covalently associated, individual Aβ(X-Y) peptides,which show fibrillary structure in the electron microscope, which bindCongo red and then exhibit birefringence under polarized light and whoseX-ray diffraction pattern is a cross-β structure. In another aspect ofthe invention, a fibril is a molecular structure obtainable by a processthat comprises the self-induced polymeric aggregation of a suitable Aβpeptide in the absence of detergents, e.g., in 0.1 M HCl, leading to theformation of aggregates of more than 24, preferably more than 100 units.This process is well known in the art. Expediently, Aβ(X-Y) fibrils areused in the form of an aqueous solution. In a particularly preferredembodiment of the invention, the aqueous fibril solution is made bydissolving the Aβ peptide in 0.1% NH₄OH, diluting it 1:4 with 20 mMNaH₂PO₄, 140 mM NaCl, pH 7.4, followed by readjusting the pH to 7.4,incubating the solution at 37° C. for 20 h, followed by centrifugationat 10000 g for 10 min and resuspension in 20 mM NaH₂PO₄, 140 mM NaCl, pH7.4.

The term “Aβ(X-Y) fibril” herein refers to a fibril consistingessentially of Aβ(X-Y) subunits, where it is preferred if on average atleast 90% of the subunits are of the Aβ(X-Y) type, more preferred, if atleast 98% of the subunits are of the Aβ(X-Y) type and, most preferred,if the content of non-Aβ(X-Y) peptides is below the detection threshold.

The present invention also relates to antibodies having a similarbinding profile to that of any one of said monoclonal antibodies, 10F4and 3C5. Antibodies having a binding profile similar to that of any oneof said monoclonal antibodies include antibodies which bind to the sameepitope as monoclonal antibody 10F4 and 3C5.

The present invention also relates to antibodies which are capable ofcompeting with at least one, preferably all, antibodies selected fromthe group consisting of 10F4 and 3C5. The term “competing antibodies”herein refers to any number of antibodies targeting the same molecularor stably but non-covalently linked supermolecular entity, preferably,the same molecule, wherein at least one is capable of specificallyreducing the measurable binding of another, preferably, by stericallyhampering the other's access to its target epitope or by inducing and/orstabilizing a conformation in the target entity that reduces thetarget's affinity for the other antibody, more preferably, by directlyblocking access to the other's target epitope by binding to an epitopein sufficiently close vicinity of the former, overlapping with theformer or identical to the former, most preferably, overlapping oridentical, in particular identical. Two epitopes are said to be“overlapping” if they share part of their chemical structures,preferably their amino acid sequences, and to be “identical” if theirchemical structures, preferably their amino acid sequences, areidentical. Thus, the present invention also relates to antibodies whosetarget epitopes are overlapping with, preferably identical to, thetarget epitope of at least one of the antibodies selected from the groupconsisting of 10F4 and 3C5. Antibodies having a similar binding profileto that of any one of said monoclonal antibodies 10F4 and 3C5 thusfurther include antibodies which comprise at least a portion of theantigen-binding moiety of any one of said monoclonal antibodies.Preferably, said portion comprises at least one complementarydetermining region (CDR) of any one of said monoclonal antibodies. Thus,according to a further particular embodiment, the present inventionrelates to antibodies comprising the amino acid sequence of the heavychain CDR3 and/or the amino acid sequence of the light chain CDR3 ofmonoclonal antibody 10F4 or 3C5, respectively. Specific examples of suchantibodies include those which also comprise the amino acid sequence ofthe heavy chain CDR2 and/or the amino acid sequence of the light chainCDR2 of monoclonal antibody 10F4 or 3C5, respectively. Even morespecifically, such antibodies include those which also comprise theamino acid sequence of the heavy chain CDR1 and/or the amino acidsequence of the light chain CDR1 of monoclonal antibody 10F4 or 3C5,respectively. In one aspect, the present invention thus relates toantibodies comprising a heavy chain wherein the CDR3, CDR2 and/or CDR1domain comprises the amino acid sequence of the heavy chain CDR3, CDR2and/or CDR1 of monoclonal antibody 10F4 or 3C5. In a further aspect, thepresent invention thus relates to antibodies comprising a light chainwherein the CDR3, CDR2 and/or CDR1 domain comprises the amino acidsequence of the light chain CDR3, CDR2 and/or CDR1, respectively, ofmonoclonal antibody 10F4 or 3C5.

In one embodiment the antibody of the invention comprises at least twovariable domain CDR sets. More preferably, the two variable domain CDRsets are selected from the group consisting of: VH 10F4 CDR Set & VL10F4 CDR Set; VH 3C5 CDR Set & VL 3C5 CDR Set (see FIGS. 7 a-7 d).

In another embodiment the antibody disclosed above further comprises ahuman acceptor framework. In a preferred embodiment, the antibody is aCDR grafted antibody. Preferably, the CDR grafted antibody comprises oneor more of the CDRs disclosed above. Preferably the CDR grafted antibodycomprises a human acceptor framework.

In a preferred embodiment the antibody is a humanized antibody.Preferably, the humanized antibody comprises one or more of the CDRsdisclosed above. More preferably, the humanized antibody comprises threeor more of the CDRs disclosed above. Most preferably, the humanizedantibody comprises six CDRs disclosed above. In a particular embodiment,the CDRs are incorporated into a human antibody variable domain of ahuman acceptor framework. Preferably, the human antibody variable domainis a consensus human variable domain. More preferably, the humanacceptor framework comprises at least one framework region amino acidsubstitution at a key residue, wherein the key residue is selected fromthe group consisting of a residue adjacent to a CDR; a glycosylationsite residue; a rare residue; a residue capable of interacting with aCDR; a canonical residue; a contact residue between heavy chain variableregion and light chain variable region; a residue within a Vernier zone;and a residue in a region that overlaps between a Chothia-definedvariable heavy chain CDR1 and a Kabat-defined first heavy chainframework. Preferably, the human acceptor framework human acceptorframework comprises at least one framework region amino acidsubstitution, wherein the amino acid sequence of the framework is atleast 65% identical to the sequence of said human acceptor framework andcomprises at least 70 amino acid residues identical to said humanacceptor framework. In yet a further aspect, the present inventionrelates to antibodies comprising both the heavy and light chain asdefined above. Preferably, the antibody comprises at least one variabledomain as described above. More preferably, the antibody comprises twovariable domains as described above, wherein said two variable domainshave amino acid sequences as noted in FIG. 7.

In another aspect, the antibodies of the present invention comprise aheavy chain constant region selected from the group consisting of IgG1,IgG2, IgG3, IgG4, IgM, IgA, IgD, IgE and human IgG1 Ala234 Ala235 mutantconstant regions. In particular, the antibodies comprise a humanconstant region. Antibodies comprising an IgG1 heavy chain constantregion are preferred.

In another embodiment the antibody is glycosylated. Preferably theglycosylation pattern is a human glycosylation pattern or aglycosylation pattern produced by any one of the eukaryotic cellsdisclosed herein, in particular CHO cells.

The present invention also relates to an antigen-binding moiety of anantibody of the present invention. Such antigen-binding moietiesinclude, but are not limited to, Fab fragments, F(ab′)₂ fragments andsingle chain Fv fragments of the antibody. Further antigen-bindingmoieties are Fab′ fragments, Fv fragments, and disulfide linked Fvfragments.

The invention also provides an isolated nucleic acid encoding any one ofthe antibodies disclosed herein. A further embodiment provides a vectorcomprising the isolated nucleic acid disclosed herein. The vector may inparticular be selected from the group consisting of pcDNA; pTT (Durocheret al., Nucleic Acids Research 2002, Vol 30, No. 2); pTT3 (pTT withadditional multiple cloning site; pEFBOS (Mizushima, S. and Nagata, S.,(1990) Nucleic acids Research Vol 18, No. 17); pBV; pJV; and pBJ.

In another aspect, a host cell is transformed with the vector disclosedherein. Preferably, the host cell is a prokaryotic cell. Morepreferably, the host cell is E. coli. In a related embodiment, the hostcell is an eukaryotic cell. Preferably, the eukaryotic cell is selectedfrom the group consisting of a protist cell, an animal cell, a plantcell and a fungal cell. More preferably, the host cell is a mammaliancell including, but not limited to, CHO and COS; or a fungal cell suchas Saccharomyces cerevisiae; or an insect cell such as Sf9.

Another aspect of the invention provides a method of producing anantibody of the invention, comprising culturing any one of the hostcells or a hybridoma disclosed herein in a culture medium underconditions suitable to produce the antibody. Another embodiment providesan antibody that is obtainable by the method disclosed herein.Antibodies of the present invention can be obtained in a manner knownper se. B lymphocytes which, in totality, contain an antibody repertoirecomposed of hundreds of billions of different antibody specificities area part of the mammalian immune system. A normal immune response to aparticular antigen means selection of one or more antibodies of saidrepertoire which specifically bind to said antigen, and the success ofan immune response is based at least partially on the ability of saidantibodies to specifically recognize (and ultimately to eliminate) thestimulating antigen and to ignore other molecules in the environment ofsaid antibodies. The usefulness of antibodies which specificallyrecognize one particular target antigen has led to the development ofmonoclonal antibody technology. Standardized hybridoma technology nowallows the production of antibodies with a single specificity for anantigen of interest. More recently, recombinant antibody techniques suchas in-vitro screening of antibody libraries have been developed. Thesetechniques likewise allow antibodies having a single specificity for anantigen of interest to be produced.

In the method of the invention, the antigen of interest may be allowedto act on the antibody repertoire either in vivo or in vitro. Accordingto one embodiment, the antigen is allowed to act on the repertoire byimmunizing an animal in vivo with said antigen. This in-vivo approachmay furthermore comprise establishing from the lymphocytes of an animala number of hybridomas and selecting a particular hybridoma whichsecretes an antibody specifically binding to said antigen. The animal tobe immunized may be, for example, a mouse, rat, rabbit, chicken, camelidor sheep or may be a transgenic version of any of the animals mentionedabove, for example, a transgenic mouse with human immunoglobulin genes,which produces human antibodies after an antigenic stimulus. Other typesof animals which may be immunized include mice with severe combinedimmunodeficiency (SCID) which have been reconstituted with humanperipheral mononuclear blood cells (chimeric hu-PBMC SCID mice) or withlymphoid cells or precursors thereof, as well as mice which have beentreated with a lethal total body irradiation, then protected againstradiation with bone marrow cells from a mouse with severe combinedimmunodeficiency (SCID) and subsequently transplanted with functionalhuman lymphocytes (the “Trimera” system). Another type of an animal tobe immunized is an animal (e.g., a mouse) in whose genome an endogenousgene encoding the antigen of interest has been switched off (knockedout), for example, by homologous recombination, so that afterimmunization with the antigen, said animal recognizes said antigen asforeign. The polyclonal or monoclonal antibodies produced by this methodare characterized and selected by using known screening methods whichinclude, but are not limited to, ELISA and dot blot techniques.

According to another embodiment, the antigen is allowed to act on theantibody repertoire in vitro by screening a recombinant antibody librarywith said antigen. The recombinant antibody library may be expressed,for example, on the surface of bacteriophages or on the surface of yeastcells or on the surface of bacterial cells. In a variety of embodiments,the recombinant antibody library is an scFv library or an Fab library,for example. According to another embodiment, antibody libraries areexpressed as RNA-protein fusions.

Another approach to producing antibodies of the invention comprises acombination of in vivo and in vitro approaches. For example, the antigenmay be allowed to act on the antibody repertoire by immunizing an animalin vivo with said antigen and then screening in vitro with said antigena recombinant antibody library prepared from lymphoid cells of saidanimal or a single domain antibody library (e.g., containing heavyand/or light chains). According to another approach, the antigen isallowed to act on the antibody repertoire by immunizing an animal invivo with said antigen and then subjecting a recombinant antibodylibrary or single domain library produced from lymphoid cells of saidanimal to affinity maturation. According to another approach, theantigen is allowed to act on the antibody repertoire by immunizing ananimal in vivo with said antigen, then selecting individualantibody-producing cells secreting an antibody of interest and obtainingfrom said selected cells cDNAs for the variable region of the heavy andlight chains (e.g., by means of PCR) and expressing said variableregions of the heavy and light chains in mammalian host cells in vitro(this being referred to as selected lymphocyte antibody method or SLAM),thereby being able to further select and manipulate the selectedantibody gene sequences. Moreover, monoclonal antibodies may be selectedby expression cloning by expressing the antibody genes for the heavy andlight chains in mammalian cells and selecting those mammalian cellswhich secrete an antibody having the desired binding affinity.

The methods of the invention for producing antibodies can be used toproduce various types of antibodies. These include monoclonal, inparticular recombinant antibodies, especially essentially humanantibodies, chimeric antibodies, humanized antibodies and CDR graftantibodies, and also antigen-binding moieties thereof.

The present invention further relates to a hybridoma that is capable ofproducing (secreting) a monoclonal antibody of the present invention.Hybridomas of the present invention include those designated by anAmerican Type Culture Collection deposit number selected from the groupconsisting of PTA-7808 and PTA-7406 and those producing monoclonalantibodies 10F4 and 3C5.

It is noted that the antibodies of the present invention may also bereactive with, i.e., bind to, Aβ forms other than the Aβ globulomersdescribed herein. These antigens may or may not be oligomeric orglobulomeric. Thus, the antigens to which the antibodies of the presentinvention bind include any Aβ form that comprises the globulomer epitopewith which the antibodies of the present invention are reactive. Such Aβforms include truncated and non-truncated Aβ(X-Y) forms (with X and Ybeing defined as above), such as Aβ(20-42), Aβ(20-40), Aβ(12-42),Aβ(12-40), Aβ(1-42), and Aβ(1-40) forms, provided that said formscomprise the globulomer epitope.

The present invention also relates to a composition comprising anantibody of the invention or an antigen-binding moiety thereof, asdefined above. According to a particular embodiment, said composition isa pharmaceutical composition which comprises the antibody of theinvention or the antigen-binding moiety and a pharmaceutical acceptablecarrier. The antibody of the invention or the antigen-binding moiety, asdefined above, is preferably capable of neutralizing, both in vitro andin vivo, the activity of Aβ globulomer or a derivative thereof to whichit binds. Said antibody or antigen-binding moiety may therefore be usedfor inhibiting the activity of said globulomer or derivative thereof,for example, in a preparation containing said globulomer or derivativethereof or in human individuals or other mammals in which saidglobulomer or derivative thereof is present.

According to one embodiment, the invention relates to a method ofinhibiting the activity of said globulomer or derivative thereof whichmethod comprises allowing an antibody of the invention or anantigen-binding moiety thereof to act on a globulomer or derivativethereof so as to inhibit the activity of said globulomer or derivativethereof. Said activity may be inhibited in vitro, for example. Forinstance, the antibody of the invention or the antigen-binding moietymay be added to a preparation such as a sample derived from a subject ora cell culture which contains or is suspected to contain said globulomeror derivative thereof, in order to inhibit the activity of saidglobulomer or derivative thereof in said sample. Alternatively, theactivity of the globulomer or derivative thereof may be inhibited in anindividual in vivo. Thus, the present invention further relates to theuse of an antibody or an antigen-binding moiety as defined above forpreparing a pharmaceutical composition for treating or preventing anamyloidosis, in particular, an amyloidosis selected from the groupconsisting of Alzheimer's disease and the amyloidosis of Down'ssyndrome. One aspect of said use of the invention is therefore a methodof treating or preventing an amyloidosis, in particular, Alzheimer'sdisease or the amyloidosis of Down's syndrome, in a subject in needthereof, which comprises administering an antibody or an antigen-bindingmoiety as defined above to the subject. Using said antibody orantigen-binding moiety for treating and especially preventing theamyloidosis, in particular, Alzheimer's disease or the amyloidosis ofDown's syndrome, is in particular for passive immunization. Accordingly,in the method of treating or preventing an amyloidosis, in particularAlzheimer's disease or the amyloidosis of Down's syndrome, in a subjectin need thereof one purpose of administering the antibody orantigen-binding moiety to the subject is passively immunizing thesubject against the amyloidosis, in particular, Alzheimer's disease orthe amyloidosis of Down's syndrome.

The antibody of the invention or the antigen-binding moiety as definedabove is preferably capable of detecting, both in vitro and in vivo, anAβ globulomer or derivative thereof to which it binds. Said antibody orthe antigen-binding moiety may therefore be used for detecting saidglobulomer or derivative thereof, for example, in a preparationcontaining said globulomer or derivative thereof or in human individualsor other mammals in which said globulomer or derivatives thereof ispresent.

According to one embodiment, the invention relates to a method ofdetecting said globulomer or derivative thereof, which method comprisesallowing an antibody of the invention or an antigen-binding moietythereof to act on a globulomer or derivative thereof so as to bind tosaid globulomer or derivative thereof (and thereby preferably forming acomplex comprising the antibody or antigen-binding moiety thereof andthe globulomer or derivative thereof). The globulomer may be detected invitro, for example. For instance, the antibody of the invention or theantigen-binding moiety may be added to a preparation, for instance, asample derived from a subject or a cell culture which contains or issuspected to contain said globulomer or derivative thereof, in order todetect said globulomer or derivative thereof in said preparation.Alternatively, the globulomer or derivative thereof may be detected inan individual in vivo. Thus, the present invention further relates tothe use of an antibody or an antigen-binding moiety as defined above forpreparing a composition for diagnosing an amyloidosis, in particularAlzheimer's disease or the amyloidosis of Down's syndrome. One aspect ofsaid use of the invention is a method of diagnosing an amyloidosis, inparticular, Alzheimer's disease or the amyloidosis of Down's syndrome,in a subject suspected of having the amyloidosis, in particularAlzheimer's disease or the amyloidosis of Down's syndrome, whichcomprises administering to the subject an antibody or an antigen-bindingmoiety as defined above and detecting the formation of a complexcomprising the antibody or the antigen-binding moiety with the antigen,the presence of the complex indicating the amyloidosis, in particularAlzheimer's disease or the amyloidosis of Down's syndrome, in thesubject. A second aspect of said use of the invention is a method ofdiagnosing an amyloidosis, in particular, Alzheimer's disease or theamyloidosis of Down's syndrome, in a subject suspect of having theamyloidosis, in particular, Alzheimer's disease or the amyloidosis ofDown's syndrome, which comprises providing a sample from the subject,contacting the sample with an antibody or an antigen-binding moiety (asdefined) above and detecting the formation of a complex comprising theantibody or the antigen-binding moiety with the antigen, the presence ofthe complex indicating the amyloidosis, in particular, Alzheimer'sdisease or the amyloidosis of Down's syndrome, in the subject.

The binding affinities of the antibodies of the invention may beevaluated by using standardized in-vitro immunoassays such as ELISA, dotblot or BIAcore analyses (Pharmacia Biosensor AB, Uppsala, Sweden andPiscataway, N.J.). For further descriptions, see Jönsson, U., et al.(1993) Ann. Biol. Clin. 51:19-26; Jönsson, U., et al. (1991)Biotechniques 11:620-627; Johnsson, B., et al. (1995) J. Mol. Recognit.8:125-131; and Johnnson, B., et al. (1991) Anal. Biochem. 198:268-277.According to a particular embodiment, the affinities defined hereinrefer to the values obtained by performing a dot blot and evaluating itby densitometry. According to a particular embodiment of the invention,determining the binding affinity by dot blot comprises the following: acertain amount of the antigen (e.g. the Aβ(X-Y) globulomer, Aβ(X-Y)monomer or Aβ(x-Y) fibrils, as defined above) or, expediently, anappropriate dilution thereof, for instance in 20 mM NaH₂PO₄, 140 mMNaCl, pH 7.4, 0.2 mg/mL BSA to an antigen concentration of, for example,100 pmol/μL, 10 pmol/μL, 1 pmol/μL, 0.1 pmol/μL and 0.01 pmol/μL, isdotted onto a nitrocellulose membrane, the membrane is then blocked withmilk to prevent unspecific binding and washed, then contacted with theantibody of interest followed by detection of the latter by means of anenzyme-conjugated secondary antibody and a colorimetric reaction; atdefined antibody concentrations, the amount of antibody bound allowsaffinity determination. Thus the relative affinity of two differentantibodies to one target, or of one antibody to two different targets,is here defined as the relation of the respective amounts oftarget-bound antibody observed with the two antibody-target combinationsunder otherwise identical dot blot conditions. Unlike a similar approachbased on Western blotting, the dot blot approach will determine anantibody's affinity to a given target in the latter's naturalconformation; unlike the ELISA approach, the dot blot approach does notsuffer from differences in the affinities between different targets andthe matrix, thereby allowing for more precise comparisons betweendifferent targets.

The term “K_(d)”, as used herein, is intended to refer to thedissociation constant of a particular antibody-antigen interaction as isknown in the art.

The antibodies of the present invention are preferably isolatedantibodies. An isolated antibody” means an antibody having the bindingaffinities as described above and which is essentially free of otherantibodies having different binding affinities. The term “essentiallyfree” here refers to an antibody preparation in which at least 95% ofthe antibodies, preferably at least 98% of the antibodies and morepreferably at least 99% of the antibodies have the desired bindingaffinity. Moreover, an isolated antibody may be substantially free ofother cellular material and/or chemicals.

The isolated antibodies of the present invention include monoclonalantibodies. A “monoclonal antibody” as used herein is intended to referto a preparation of antibody molecules, antibodies which share a commonheavy chain and common light chain amino acid sequence, in contrast with“polyclonal” antibody preparations which contain a mixture of antibodiesof different amino acid sequence. Monoclonal antibodies can be generatedby several novel technologies like phage, bacteria, yeast or ribosomaldisplay, as well as by classical methods exemplified byhybridoma-derived antibodies (e.g., an antibody secreted by a hybridomaprepared by hybridoma technology, such as the standard Kohler andMilstein hybridoma methodology ((1975) Nature 256:495-497). Thus, anon-hybridoma-derived antibody with uniform sequence is still referredto as a monoclonal antibody herein although it may have been obtained bynon-classical methodologies, and the term “monoclonal” is not restrictedto hybridoma-derived antibodies but used to refer to all antibodiesderived from one nucleic acid clone. Thus, the monoclonal antibodies ofthe present invention include recombinant antibodies. The term“recombinant” as used herein refers to any artificial combination of twootherwise separated segments of sequence, e.g., by chemical synthesis orby the manipulation of isolated segments of nucleic acids by geneticengineering techniques. In particular, the term “recombinant antibody”refers to antibodies which are produced, expressed, generated orisolated by recombinant means, such as antibodies which are expressedusing a recombinant expression vector transfected into a host cell;antibodies isolated from a recombinant combinatorial antibody library;antibodies isolated from an animal (e.g. a mouse) which is transgenicdue to human immunoglobulin genes (see, for example, Taylor, L. D., etal. (1992) Nucl. Acids Res. 20:6287-6295); or antibodies which areproduced, expressed, generated or isolated in any other way in whichparticular immunoglobulin gene sequences (such as human immunoglobulingene sequences) are assembled with other DNA sequences. Recombinantantibodies include, for example, chimeric, CDR graft and humanizedantibodies. The person skilled in the art will be aware that expressionof a conventional hybridoma-derived monoclonal antibody in aheterologous system will require the generation of a recombinantantibody even if the amino acid sequence of the resulting antibodyprotein is not changed or intended to be changed.

In a particular embodiment of the invention, the antibody is a humanizedantibody. According to a multiplicity of embodiments, the antibody maycomprise an amino acid sequence derived entirely from a single species,such as a human antibody or a mouse antibody. According to otherembodiments, the antibody may be a chimeric antibody or a CDR graftantibody or another form of a humanized antibody.

The term “antibody” is intended to refer to immunoglobulin moleculesconsisting of 4 polypeptide chains, two heavy (H) chains and two light(L) chains. The chains are usually linked to one another via disulfidebonds. Each heavy chain is composed of a variable region of said heavychain (abbreviated here as HCVR or VH) and a constant region of saidheavy chain. The heavy chain constant region consists of three domainsCH1, CH2 and CH3. Each light chain is composed of a variable region ofsaid light chain (abbreviated here as LCVR or VL) and a constant regionof said light chain. The light chain constant region consists of a CLdomain. The VH and VL regions may be further divided into hypervariableregions referred to as complementarity-determining regions (CDRs) andinterspersed with conserved regions referred to as framework regions(FR). Each VH and VL region thus consists of three CDRs and four FRswhich are arranged from the N terminus to the C terminus in thefollowing order: FR1, CDR1, FR2, CDR2, FR3, CDR3, FR4. This structure iswell known to those skilled in the art.

The term “antigen-binding moiety” of an antibody (or simply “antibodymoiety”) refers to one or more fragments of an antibody of theinvention, said fragment(s) still having the binding affinities asdefined above. Fragments of a complete antibody have been shown to beable to carry out the antigen-binding function of an antibody. Inaccordance with the term “antigen-binding moiety” of an antibody,examples of binding fragments include (i) an Fab fragment, i.e. amonovalent fragment composed of the VL, VH, CL and CH1 domains; (ii) anF(ab′)₂ fragment, i.e. a bivalent fragment comprising two Fab fragmentslinked to one another in the hinge region via a disulfide bridge; (iii)an Fd fragment composed of the VH and CH1 domains; (iv) an Fv fragmentcomposed of the FL and VH domains of a single arm of an antibody; (v) adAb fragment (Ward et al., (1989) Nature 341:544-546) consisting of a VHdomain or of VH, CH1, CH2, DH3, or VH, CH2, CH3; and (vi) an isolatedcomplementarity-determining region (CDR). Although the two domains ofthe Fv fragment, namely VL and VH, are encoded by separate genes, theymay further be linked to one another using a synthetic linker, e.g. apoly-G₄S amino acid sequence, and recombinant methods, making itpossible to prepare them as a single protein chain in which the VL andVH regions combine in order to form monovalent molecules (known assingle chain Fv (ScFv); see, for example, Bird et al. (1988) Science242:423-426; and Huston et al. (1988) Proc. Natl. Acad. Sci. USA85:5879-5883). The term “antigen-binding moiety” of an antibody is alsointended to comprise such single chain antibodies. Other forms of singlechain antibodies such as “diabodies” are likewise included here.Diabodies are bivalent, bispecific antibodies in which VH and VL domainsare expressed on a single polypeptide chain, but using a linker which istoo short for the two domains being able to combine on the same chain,thereby forcing said domains to pair with complementary domains of adifferent chain and to form two antigen-binding sites (see, for example,Holliger, P., et al. (1993) Proc. Natl. Acad. Sci. USA 90:6444-6448;Poljak, R. J., et al. (1994) Structure 2:1121-1123). An immunoglobulinconstant domain refers to a heavy or light chain constant domain. HumanIgG heavy chain and light chain constant domain amino acid sequences areknown in the art.

Furthermore, an antibody of the present invention or antigen-bindingmoiety thereof may be part of a larger immunoadhesion molecule formed bycovalent or noncovalent association of said antibody or antibody moietywith one or more further proteins or peptides. Relevant to suchimmunoadhesion molecules are the use of the streptavidin core region inorder to prepare a tetrameric scFv molecule (Kipriyanov, S. M., et al.(1995) Human Antibodies and Hybridomas 6:93-101) and the use of acystein residue, a marker peptide and a C-terminal polyhistidinyl, e.g.hexahistidinyl, tag in order to produce bivalent and biotinylated scFvmolecules (Kipriyanov, S. M., et al. (1994) Mol. Immunol. 31:1047-1058).

The term “human antibody” refers to antibodies whose variable andconstant regions correspond to or are derived from immunoglobulinsequences of the human germ line, as described, for example, by Kabat etal. (see Kabat, et al. (1991) Sequences of Proteins of ImmunologicalInterest, Fifth Edition, U.S. Department of Health and Human Services,NIH Publication No. 91-3242). However, the human antibodies of theinvention may contain amino acid residues not encoded by human germ lineimmunoglobulin sequences (for example mutations which have beenintroduced by random or site-specific mutagenesis in vitro or by somaticmutation in vivo), for example in the CDRs, and in particular in CDR3.Recombinant human antibodies of the invention have variable regions andmay also contain constant regions derived from immunoglobulin sequencesof the human germ line (see Kabat, E. A., et al. (1991) Sequences ofProteins of Immunological Interest, Fifth Edition, U.S. Department ofHealth and Human Services, NIH

Publication No. 91-3242). According to particular embodiments, however,such recombinant human antibodies are subjected to in-vitro mutagenesis(or to a somatic in-vivo mutagenesis, if an animal is used which istransgenic due to human Ig sequences) so that the amino acid sequencesof the VH and VL regions of the recombinant antibodies are sequenceswhich although related to or derived from VH and VL sequences of thehuman germ line, do not naturally exist in vivo within the humanantibody germ line repertoire. According to particular embodiments,recombinant antibodies of this kind are the result of selectivemutagenesis or back mutation or of both. Preferably, mutagenesis leadsto an affinity to the target which is greater, and/or an affinity tonon-target structures which is smaller than that of the parent antibody.

The term “chimeric antibody” refers to antibodies which containsequences for the variable region of the heavy and light chains from onespecies and constant region sequences from another species, such asantibodies having murine heavy and light chain variable regions linkedto human constant regions.

The term “CDR-grafted antibody” refers to antibodies which compriseheavy and light chain variable region sequences from one species but inwhich the sequences of one or more of the CDR regions of VH and/or VLare replaced with CDR sequences of another species, such as antibodieshaving murine heavy and light chain variable regions in which one ormore of the murine CDRs (e.g., CDR3) has been replaced with human CDRsequences.

The term “humanized antibody” refers to antibodies which containsequences of the variable region of heavy and light chains from anonhuman species (e.g. mouse, rat, rabbit, chicken, camelid, sheep orgoat) but in which at least one part of the VH and/or VL sequence hasbeen altered in order to be more “human-like”, i.e. to be more similarto variable sequences of the human germ line. One type of a humanizedantibody is a CDR graft antibody in which human CDR sequences have beeninserted into nonhuman VH and VL sequences to replace the correspondingnonhuman CDR sequences.

The terms “Kabat numbering”, “Kabat definitions” and “Kabat labeling”are used interchangeably herein. These terms, which are recognized inthe art, refer to a system of numbering amino acid residues which aremore variable (i.e. hypervariable) than other amino acid residues in theheavy and light chain variable regions of an antibody, or an antigenbinding portion thereof (Kabat et al. (1971) Ann. NY Acad, Sci.190:382-391 and Kabat, E. A., et al. (1991) Sequences of Proteins ofImmunological Interest, Fifth Edition, U.S. Department of Health andHuman Services, NIH Publication No. 91-3242).

As used herein, the terms “acceptor” and “acceptor antibody” refer tothe antibody or nucleic acid sequence providing or encoding at least80%, at least 85%, at least 90%, at least 95%, at least 98% or 100% ofthe amino acid sequences of one or more of the framework regions. Insome embodiments, the term “acceptor” refers to the antibody amino acidor nucleic acid sequence providing or encoding the constant region(s).In yet another embodiment, the term “acceptor” refers to the antibodyamino acid or nucleic acid sequence providing or encoding one or more ofthe framework regions and the constant region(s). In a specificembodiment, the term “acceptor” refers to a human antibody amino acid ornucleic acid sequence that provides or encodes at least 80%, preferably,at least 85%, at least 90%, at least 95%, at least 98%, or 100% of theamino acid sequences of one or more of the framework regions. Inaccordance with this embodiment, an acceptor may contain at least 1, atleast 2, at least 3, least 4, at least 5, or at least 10 amino acidresidues not occurring at one or more specific positions of a humanantibody. An acceptor framework region and/or acceptor constantregion(s) may be, e.g., derived or obtained from a germline antibodygene, a mature antibody gene, a functional antibody (e.g., antibodieswell-known in the art, antibodies in development, or antibodiescommercially available).

As used herein, the term “CDR” refers to the complementarity determiningregion within antibody variable sequences. There are three CDRs in eachof the variable regions of the heavy chain and of the light chain, whichare designated CDR1, CDR2 and CDR3, for each of the variable regions.The term “CDR set” as used herein refers to a group of three CDRs thatoccur in a single variable region capable of binding the antigen. Theexact boundaries of these CDRs have been defined differently accordingto different systems. The system described by Kabat (Kabat et al.,Sequences of Proteins of Immunological Interest (National Institutes ofHealth, Bethesda, Md. (1987) and (1991)) not only provides anunambiguous residue numbering system applicable to any variable regionof an antibody, but also provides precise residue boundaries definingthe three CDRs. These CDRs may be referred to as Kabat CDRs. Chothia andcoworkers (Chothia & Lesk, J. Mol. Biol. 196:901-917 (1987) and Chothiaet al., Nature 342:877-883 (1989)) found that certain sub-portionswithin Kabat CDRs adopt nearly identical peptide backbone conformations,in spite of great diversity at the level of amino acid sequence. Thesesub-portions were designated as L1, L2 and L3 or H1, H2 and H3 where the“L” and the “H” designates the light chain and the heavy chains regions,respectively. These regions may be referred to as Chothia CDRs, whichhave boundaries that overlap with Kabat CDRs. Other boundaries definingCDRs overlapping with the Kabat CDRs have been described by Padlan(FASEB J. 9:133-139 (1995)) and MacCallum (J Mol Biol 262(5):732-45(1996)). Still other CDR boundary definitions may not strictly followone of the above systems, but will nonetheless overlap with the KabatCDRs, although they may be shortened or lengthened in light ofprediction or experimental findings that particular residues or groupsof residues or even entire CDRs do not significantly impact antigenbinding. The methods used herein may utilize CDRs defined according toany of these systems, although preferred embodiments use Kabat orChothia defined CDRs.

As used herein, the term “canonical” residue refers to a residue in aCDR or framework that defines a particular canonical CDR structure asdefined by Chothia et al. (J. Mol. Biol. 196:901-907 (1987); Chothia etal., J. Mol. Biol. 227:799 (1992), both are incorporated herein byreference). According to Chothia et al., critical portions of the CDRsof many antibodies have nearly identical peptide backbone confirmationsdespite great diversity at the level of amino acid sequence. Eachcanonical structure specifies primarily a set of peptide backbonetorsion angles for a contiguous segment of amino acid residues forming aloop.

As used herein, the terms “donor” and “donor antibody” refer to anantibody providing one or more CDRs. In a preferred embodiment, thedonor antibody is an antibody from a species different from the antibodyfrom which the framework regions are obtained or derived. In the contextof a humanized antibody, the term “donor antibody” refers to a non-humanantibody providing one or more CDRs.

As used herein, the term “framework” or “framework sequence” refers tothe remaining sequences of a variable region minus the CDRs. Because theexact definition of a CDR sequence can be determined using differentsystems, the meaning of a framework sequence is subject tocorrespondingly different interpretations. The six CDRs (CDR-L1, -L2,and -L3 of light chain and CDR-H1, -H2, and -H3 of heavy chain) alsodivide the framework regions on the light chain and the heavy chain intofour sub-regions (FR1, FR2, FR3 and FR4) on each chain, in which CDR1 ispositioned between FR1 and FR2, CDR2 between FR2 and FR3, and CDR3between FR3 and FR4. Without specifying the particular sub-regions asFR1, FR2, FR3 or FR4, a framework region, as referred by others,represents the combined FR's within the variable region of a single,naturally occurring immunoglobulin chain. As used herein, a FRrepresents one of the four sub-regions, and FRs represents two or moreof the four sub-regions constituting a framework region. Human heavychain and light chain acceptor sequences are known in the art.

As used herein, the term “germline antibody gene” or “gene fragment”refers to an immunoglobulin sequence encoded by non-lymphoid cells thathave not undergone the maturation process that leads to geneticrearrangement and mutation for expression of a particularimmunoglobulin. (See, e.g., Shapiro et al., Crit. Rev. Immunol. 22(3):183-200 (2002); Marchalonis et al., Adv Exp Med. Biol. 484:13-30(2001)). One of the advantages provided by various embodiments of thepresent invention stems from the finding that germline antibody genesare more likely than mature antibody genes are to conserve essentialamino acid sequence structures characteristic of individuals in thespecies, hence less likely to be recognized as non-self when used inthat species.

As used herein, the term “key” residues refers to certain residueswithin the variable region that have more impact on the bindingspecificity and/or affinity of an antibody, in particular a humanizedantibody. A key residue includes, but is not limited to, one or more ofthe following: a residue that is adjacent to a CDR, a potentialglycosylation site (which can be either N- or O-glycosylation site), arare residue, a residue capable of interacting with the antigen, aresidue capable of interacting with a CDR, a canonical residue, acontact residue between heavy chain variable region and light chainvariable region, a residue within the Vernier zone, and a residue in theregion that overlaps between the Chothia definition of a variable heavychain CDR1 and the Kabat definition of the first heavy chain framework.

As used herein, the term “humanized antibody” specifically refers to anantibody or a variant, derivative, analog or fragment thereof whichimmunospecifically binds to an antigen of interest and which comprises aframework (FR) region having substantially the amino acid sequence of ahuman antibody and a complementary determining region (CDR) havingsubstantially the amino acid sequence of a non-human antibody. As usedherein, the term “substantially” in the context of a CDR refers to a CDRhaving an amino acid sequence at least 80%, preferably at least 85%, atleast 90%, at least 95%, at least 98% or at least 99% identical to theamino acid sequence of a non-human antibody CDR. A humanized antibodycomprises substantially all of at least one, and typically two, variabledomains (Fab, Fab′, F(ab′)₂, FabC, Fv) in which all or substantially allof the CDR regions correspond to those of a non-human immunoglobulin(i.e., donor antibody) and all or substantially all of the frameworkregions are those of a human immunoglobulin consensus sequence.Preferably, a humanized antibody also comprises at least a portion of animmunoglobulin constant region (Fc), typically that of a humanimmunoglobulin. In some embodiments, a humanized antibody contains boththe light chain as well as at least the variable domain of a heavychain. The antibody also may include the CH1, hinge, CH2, CH3, and CH4regions of the heavy chain. In some embodiments, a humanized antibodyonly contains a humanized light chain. In some embodiments, a humanizedantibody only contains a humanized heavy chain. In specific embodiments,a humanized antibody only contains a humanized variable domain of alight chain and/or humanized heavy chain. The humanized antibody can beselected from any class of immunoglobulins, including IgM, IgG, IgD, IgAand IgE, and any subclass, including without limitation IgG 1, IgG2,IgG3 and IgG4. The framework and CDR regions of a humanized antibodyneed not correspond precisely to the parental sequences, e.g., the donorantibody CDR or the consensus framework may be mutagenized bysubstitution, insertion and/or deletion of at least one amino acidresidue so that the CDR or framework residue at that site does notcorrespond exactly to either the donor antibody or the consensusframework. In a preferred embodiment, such mutations, however, will notbe extensive. Usually, at least 80%, preferably at least 85%, morepreferably at least 90%, and most preferably at least 95% of thehumanized antibody residues will correspond to those of the parental FRand CDR sequences. As used herein, the term “consensus framework” refersto the framework region in the consensus immunoglobulin sequence. Asused herein, the term “consensus immunoglobulin sequence” refers to thesequence formed from the most frequently occurring amino acids (ornucleotides) in a family of related immunoglobulin sequences (See e.g.,Winnaker, From Genes to Clones, Verlagsgesellschaft, Weinheim, Germany1987). In a family of immunoglobulins, each position in the consensussequence is occupied by the amino acid occurring most frequently at thatposition in the family. Where two amino acids occur equally frequently,either can be included in the consensus sequence.

As used herein, “Vernier” zone refers to a subset of framework residuesthat may adjust CDR structure and fine-tune the fit to antigen asdescribed by Foote and Winter (1992, J. Mol. Biol. 224:487-499, which isincorporated herein by reference). Vernier zone residues form a layerunderlying the CDRs and may impact on the structure of CDRs and theaffinity of the antibody.

The term “epitope” includes any polypeptide determinant capable ofspecific binding to an immunoglobulin. In certain embodiments, epitopedeterminants include chemically active surface groupings of moleculessuch as amino acids, sugar side chains, phosphoryl, or sulfonyl, and, incertain embodiments, may have specific three dimensional structuralcharacteristics, and/or specific charge characteristics. An epitope is aregion of an antigen that is bound by an antibody. In certainembodiments, an antibody is said to specifically bind an antigen when itpreferentially recognizes its target antigen in a complex mixture ofproteins and/or macromolecules.

The term “polynucleotide” as referred to herein means a polymeric formof two or more nucleotides, either ribonucleotides or deoxynucleotidesor a modified form of either type of nucleotide. The term includessingle and double stranded forms of DNA but preferably isdouble-stranded DNA.

The term “isolated polynucleotide” as used herein shall mean apolynucleotide (e.g., of genomic, cDNA, or synthetic origin, or anycombination thereof) that, by virtue of its origin, the “isolatedpolynucleotide” is not associated with all or a portion of apolynucleotide with which the “isolated polynucleotide” is found innature; is operably linked to a polynucleotide that it is not linked toin nature; or does not occur in nature as part of a larger sequence.

The term “vector”, as used herein, is intended to refer to a nucleicacid molecule capable of transporting another nucleic acid to which ithas been linked. One type of vector is a “plasmid”, which refers to acircular double stranded DNA into which additional DNA segments may beligated. Another type of vector is a viral vector, wherein additionalDNA segments may be ligated into the viral genome. Certain vectors arecapable of autonomous replication in a host cell into which they areintroduced (e.g., bacterial vectors having a bacterial origin ofreplication and episomal mammalian vectors). Other vectors (e.g.,non-episomal mammalian vectors) can be integrated into the genome of ahost cell upon introduction into the host cell, and thereby arereplicated along with the host genome. Moreover, certain vectors arecapable of directing the expression of genes to which they areoperatively linked. Such vectors are referred to herein as “recombinantexpression vectors” (or simply, “expression vectors”). In general,expression vectors of utility in recombinant DNA techniques are often inthe form of plasmids. In the present specification, “plasmid” and“vector” may be used interchangeably as the plasmid is the most commonlyused form of vector. However, the invention is intended to include suchother forms of expression vectors, such as viral vectors (e.g.,replication defective retroviruses, adenoviruses and adeno-associatedviruses), which serve equivalent functions.

The term “operably linked” refers to a juxtaposition wherein thecomponents described are in a relationship permitting them to functionin their intended manner. A control sequence “operably linked” to acoding sequence is connected in such a way that expression of the codingsequence is achieved under conditions compatible with the controlsequences. “Operably linked” sequences include both expression controlsequences that are contiguous with the gene of interest and expressioncontrol sequences that act in trans or at a distance to control the geneof interest. The term “expression control sequence” as used hereinrefers to polynucleotide sequences which are necessary to effect theexpression and processing of coding sequences to which they are ligated.Expression control sequences include appropriate transcriptioninitiation, termination, promoter and enhancer sequences; efficient RNAprocessing signals such as splicing and polyadenylation signals;sequences that stabilize cytoplasmic mRNA; sequences that enhancetranslation efficiency (i.e., Kozak consensus sequence); sequences thatenhance protein stability; and when desired, sequences that enhanceprotein secretion. The nature of such control sequences differsdepending upon the host organism; in prokaryotes, such control sequencesgenerally include promoter, ribosomal binding site, and transcriptiontermination sequence; in eukaryotes, generally, such control sequencesinclude promoters and transcription termination sequence. The term“control sequences” is intended to include components whose presence isessential for expression and processing, and can also include additionalcomponents whose presence is advantageous, for example, leader sequencesand fusion partner sequences.

“Transformation”, as defined herein, refers to any process by whichexogenous DNA enters a host cell. Transformation may occur under naturalor artificial conditions using various methods well known in the art.Transformation may rely on any known method for the insertion of foreignnucleic acid sequences into a prokaryotic or eukaryotic host cell. Themethod is selected based on the host cell being transformed and mayinclude, but is not limited to, viral infection, electroporation,lipofection, and particle bombardment. Such “transformed” cells includestably transformed cells in which the inserted DNA is capable ofreplication either as an autonomously replicating plasmid or as part ofthe host chromosome. They also include cells which transiently expressthe inserted DNA or RNA for limited periods of time.

The term “recombinant host cell” (or simply “host cell”), as usedherein, is intended to refer to a cell into which exogenous DNA has beenintroduced. It should be understood that such terms are intended torefer not only to the particular subject cell, but, also to the progenyof such a cell. Because certain modifications may occur in succeedinggenerations due to either mutation or environmental influences, suchprogeny may not, in fact, be identical to the parent cell, but are stillincluded within the scope of the term “host cell” as used herein.Preferably host cells include prokaryotic and eukaryotic cells selectedfrom any of the kingdoms of life. Preferred eukaryotic cells includeprotist, fungal, plant and animal cells. Most preferably host cellsinclude but are not limited to the prokaryotic cell line E. coli;mammalian cell lines CHO, HEK 293 and COS; the insect cell line Sf9; andthe fungal cell Saccharomyces cerevisiae.

Standard techniques may be used for recombinant DNA, oligonucleotidesynthesis, and tissue culture and transformation (e.g., electroporation,lipofection). Enzymatic reactions and purification techniques may beperformed according to manufacturer's specifications or as commonlyaccomplished in the art or as described herein. The foregoing techniquesand procedures may be generally performed according to conventionalmethods well known in the art and as described in various general andmore specific references that are cited and discussed throughout thepresent specification. See e.g., Sambrook et al., Molecular Cloning: ALaboratory Manual (2d ed., Cold Spring Harbor Laboratory Press, ColdSpring Harbor, N.Y. (1989)), which is incorporated herein by referencefor any purpose.

“Transgenic organism”, as known in the art and as used herein, refers toan organism having cells that contain a transgene, wherein the transgeneintroduced into the organism (or an ancestor of the organism) expressesa polypeptide not naturally expressed in the organism. A “transgene” isa DNA construct which is stably and operably integrated into the genomeof a cell from which a transgenic organism develops, directing theexpression of an encoded gene product in one or more cell types ortissues of the transgenic organism.

Methods of producing antibodies of the invention are described below. Adistinction is made here between in-vivo approaches, in-vitro approachesor a combination of both.

In-Vivo Approaches:

Depending on the type of the desired antibody, various host animals maybe used for in-vivo immunization. A host expressing itself an endogenousversion of the antigen of interest may be used. Alternatively, it ispossible to use a host which has been made deficient in an endogenousversion of the antigen of interest. For example, mice which had beenmade deficient in a particular endogenous protein via homologousrecombination at the corresponding endogenous gene (i.e., knockout mice)have been shown to generate a humoral response to the protein with whichthey have been immunized and therefore to be able to be used forproduction of high-affinity monoclonal antibodies to the protein (see,for example, Roes, J. et al. (1995) J. Immunol. Methods 183:231-237;Lunn, M. P. et al. (2000) J. Neurochem. 75:404-412).

A multiplicity of nonhuman mammals are suitable hosts for antibodyproduction in order to produce nonhuman antibodies of the invention.They include, for example, mice, rats, chickens, camelids, rabbits,sheep and goats (and knockout versions thereof), although preference isgiven to mice for the production of hybridoma. Furthermore, a nonhumanhost animal expressing a human antibody repertoire may be used forproducing essentially human antibodies to a human antigen with dualspecificity. Nonhuman animals of this kind include transgenic animals(e.g., mice) bearing human immunoglobulin transgenes (chimeric hu-PBMCSCID mice) and human/mouse irradiation chimeras which are described inmore detail below.

According to one embodiment, the animal immunized is a nonhuman mammal,preferably a mouse, which is transgenic due to human immunoglobulingenes so that said nonhuman mammal makes human antibodies upon antigenicstimulation. Typically, immunoglobulin transgenes for heavy and lightchains with human germ line configuration are introduced into suchanimals which have been altered such that their endogenous heavy andlight chain loci are inactive. If such animals are stimulated withantigen (e.g., with a human antigen), antibodies derived from the humanimmunoglobulin sequences (human antibodies) are produced. It is possibleto make from the lymphocytes of such animals human monoclonal antibodiesby means of standardized hybridoma technology. For a further descriptionof transgenic mice with human immunoglobulins and their use in theproduction of human antibodies, see, for example, U.S. Pat. No.5,939,598, WO 96/33735, WO 96/34096, WO 98/24893 and WO 99/53049(Abgenix Inc.), and U.S. Pat. No. 5,545,806, U.S. Pat. No. 5,569,825,U.S. Pat. No. 5,625,126, U.S. Pat. No. 5,633,425, U.S. Pat. No.5,661,016, U.S. Pat. No. 5,770,429, U.S. Pat. No. 5,814,318, U.S. Pat.No. 5,877,397 and WO 99/45962 (Genpharm Inc.); see also MacQuitty, J. J.and Kay, R. M. (1992) Science 257:1188; Taylor, L. D. et al. (1992)Nucleic Acids Res. 20:6287-6295; Lonberg, N. et al. (1994) Nature368:856-859; Lonberg, N. and Huszar, D. (1995) Int. Rev. Immunol.13:65-93; Harding, F. A. and Lonberg, N. (1995) Ann. N.Y. Acad. Sci.764:536-546; Fishwild, D. M. et al. (1996) Nature Biotechnology14:845-851; Mendez, M. J. et al. (1997) Nature Genetics 15:146-156;Green, L. L. and Jakobovits, A. (1998) J. Exp. Med. 188:483-495; Green,L. L. (1999) J. Immunol. Methods 231:11-23; Yang, X. D. et al. (1999) J.Leukoc. Biol. 66:401-410; Gallo, M. L. et al. (2000) Eur. J. Immunol.30:534-540.

According to another embodiment, the animal which is immunized may be amouse with severe combined immunodeficiency (SCID), which has beenreconstituted with human peripheral mononuclear blood cells or lymphoidcells or precursors thereof. Such mice which are referred to as chimerichu-PBMC SCID mice produce human immunoglobulin responses upon antigenicstimulation, as has been proved. For a further description of these miceand of their use for generating antibodies, see, for example, Leader, K.A. et al. (1992) Immunology 76:229-234; Bombil, F. et al. (1996)Immunobiol. 195:360-375; Murphy, W. J. et al. (1996) Semin. Immunol.8:233-241; Herz, U. et al. (1997) Int. Arch. Allergy Immunol.113:150-152; Albert, S. E. et al. (1997) J. Immunol. 159:1393-1403;Nguyen, H. et al. (1997) Microbiol. Immunol. 41:901-907; Arai, K. et al.(1998) J. Immunol. Methods 217:79-85; Yoshinari, K. and Arai, K. (1998)Hybridoma 17:41-45; Hutchins, W. A. et al. (1999) Hybridoma 18:121-129;Murphy, W. J. et al. (1999) Clin. Immunol. 90:22-27; Smithson, S. L. etal. (1999) Mol. Immunol. 36:113-124; Chamat, S. et al. (1999) J. Infect.Diseases 180:268-277; and Heard, C. et al. (1999) Molec. Med. 5:35-45.

According to another embodiment, the animal which is immunized is amouse which has been treated with a lethal does of total bodyirradiation, then protected from radiation with bone marrow cells frommice with severe combined immunodeficiency (SCID) and subsequentlytransplanted with functional human lymphocytes. This type of chimera,referred to as the Trimera system, is used in order to produce humanmonoclonal antibodies by immunizing said mice with the antigen ofinterest and then producing monoclonal antibodies by using standardizedhybridoma technology. For a further description of these mice and oftheir use for generating antibodies, see, for example, Eren, R. et al.(1998) Immunology 93:154-161; Reisner, Y. and Dagan, S. (1998) TrendsBiotechnol. 16:242-246; Ilan, E. et al. (1999) Hepatology 29:553-562;and Bocher, W. O. et al. (1999) Immunology 96:634-641.

Starting from the in-vivo generated antibody-producing cells, monoclonalantibodies may be produced by means of standardized techniques such asthe hybridoma technique originally described by Kohler and Milstein(1975, Nature 256:495-497) (see also Brown et al. (1981) J. Immunol.127:539-46; Brown et al. (1980) J Biol Chem 255:4980-83; Yeh et al.(1976) PNAS 76:2927-31; and Yeh et al. (1982) Int. J. Cancer 29:269-75).The technology of producing monoclonal antibody hybridomas issufficiently known (see generally R. H. Kenneth, in MonoclonalAntibodies: A New Dimension In Biological Analyses, Plenum PublishingCorp., New York, N.Y. (1980); E. A. Lerner (1981) Yale J. Biol. Med.,54:387-402; M. L. Gefter et al. (1977) Somatic Cell Genet., 3:231-36).Briefly, an immortalized cell line (typically a myeloma) is fused withlymphocytes (typically splenocytes or lymph node cells or peripheralblood lymphocytes) of a mammal immunized with the Aβ globulomer of theinvention or derivative thereof, and the culture supernatants of theresulting hybridoma cells are screened in order to identify a hybridomawhich produces a monoclonal antibody of the present invention. Any ofthe many well known protocols for fusing lymphocytes and immortalizedcell lines can be applied for this purpose (see also G. Galfre et al.(1977) Nature 266:550-52; Gefter et al. Somatic Cell Genet., citedsupra; Lerner, Yale J. Biol. Med., cited supra; Kenneth, MonoclonalAntibodies, cited supra). Moreover, the skilled worker will appreciatethat there are diverse variations of such methods, which are likewiseuseful. Typically, the immortalized cell line (e.g., a myeloma cellline) is derived from the same mammalian species as the lymphocytes. Forexample, murine hybridomas may be established by fusing lymphocytes froma mouse immunized with an immunogenic preparation of the invention withan immortalized mouse cell line. Preferred immortalized cell lines aremouse myeloma cell lines which are sensitive to culture mediumcontaining hypoxanthine, aminopterine and thymidine (HAT medium). Any ofa number of myeloma cell lines may be used by default as fusion partner,for example the P3-NS1/1-Ag4-1, P3-x63-Ag8.653 or Sp2/O-Ag14 myelomalines. These myeloma cell lines are available from the American TypeCulture Collection (ATCC), Manassas, Va. Typically, HAT-sensitive mousemyeloma cells are fused to mouse splenocytes using polyethylene glycol(PEG). Hybridoma cells resulting from the fusion are then selected usingHAT medium, thereby killing unfused and unproductively fused myelomacells (unfused splenocytes die after several days because they are nottransformed). Hybridoma cells producing monoclonal antibodies of theinvention are identified by screening the hybridoma culture supernatantsfor such antibodies, for example, by using a dot blot assay in order toselect those antibodies which have the binding affinities as definedabove. The monoclonal antibodies 10F4 and 3C5 all have been generatedusing the above-described in-vivo approach and thereof are obtainablefrom a hybridoma as defined herein.

Likewise, said hybridoma can be used as a source of nucleic acidencoding light and/or heavy chains in order to recombinantly produceantibodies of the present invention, as is described below in furtherdetail.

In-Vitro Approaches:

As an alternative to producing antibodies of the invention byimmunization and selection, antibodies of the invention may beidentified and isolated by screening recombinant combinatorialimmunoglobulin libraries to thereby isolate immunoglobulin librarymembers which have the required binding affinity. Kits for generatingand screening display libraries are commercially available (e.g. thePharmacia Recombinant Phage Antibody System, catalog No. 27-9400-01; andthe Stratagene SurfZAP® Phage Display Kit, catalog No. 240612). In manyembodiments, the display library is an scFv library or an Fab library.The phage display technique for screening recombinant antibody librarieshas been adequately described. Examples of methods and compounds whichcan be used particularly advantageously for generating and screeningantibody display libraries can be found, for example, in McCafferty etal. WO 92/01047, U.S. Pat. No. 5,969,108 and EP 589 877 (describes inparticular scFv display), Ladner et al. U.S. Pat. No. 5,223,409, U.S.Pat. No. 5,403,484, U.S. Pat. No. 5,571,698, U.S. Pat. No. 5,837,500 andEP 436 597 (describes pIII fusion, for example); Dower et al. WO91/17271, U.S. Pat. No. 5,427,908, U.S. Pat. No. 5,580,717 and EP 527839 (describes in particular Fab display); Winter et al. InternationalPublication WO 92/20791 and EP 368,684 (describes in particular thecloning of sequences for variable immunoglobulin domains); Griffiths etal., U.S. Pat. No. 5,885,793 and EP 589 877 (describes in particularisolation of human antibodies to human antigens by using recombinantlibraries); Garrard et al. WO 92/09690 (describes in particular phageexpression techniques); Knappik et al. WO 97/08320 (describes the humanrecombinant antibody library HuCal); Salfeld et al. WO 97/29131,(describes production of a recombinant human antibody to a human antigen(human tumor necrosis factor alpha) and also in-vitro affinitymaturation of the recombinant antibody) and Salfeld et al., U.S.Provisional Patent Application No. 60/126,603 and the patentapplications based hereupon (likewise describes production ofrecombinant human antibodies to human antigen (human interleukin-12),and also in-vitro affinity maturation of the recombinant antibody).

Further descriptions of screenings of recombinant antibody libraries canbe found in scientific publications such as Fuchs et al. (1991)Bio/Technology 9:1370-1372; Hay et al. (1992) Hum Antibod Hybridomas3:81-85; Huse et al. (1989) Science 246:1275-1281; Griffiths et al.(1993) EMBO J. 12:725-734; Hawkins et al. (1992) J Mol Biol 226:889-896;Clarkson et al. (1991) Nature 352:624-628; Gram et al. (1992) PNAS89:3576-3580; Garrard et al. (1991) Bio/Technology 9:1373-1377;Hoogenboom et al. (1991) Nuc Acid Res 19:4133-4137; Barbas et al. (1991)PNAS 88:7978-7982; McCafferty et al. Nature (1990) 348:552-554; andKnappik et al. (2000) J. Mol. Biol. 296:57-86.

As an alternative to using bacteriophage display systems, recombinantantibody libraries may be expressed on the surface of yeast cells or ofbacterial cells. WO 99/36569 describes methods of preparing andscreening libraries expressed on the surface of yeast cells. WO 98/49286describes in more detail methods of preparing and screening librariesexpressed on the surface of bacterial cells. In all in vitro approaches,a selection process for enriching recombinant antibodies with thedesired properties form an integral part of the process, which isgenerally referred to as “panning” and often takes the form of affinitychromatography over columns to whose matrix the target structure hasbeen attached. Promising candidate molecules are then subjected toindividual determination of their absolute and/or relative affinities,preferably by means of a standardized dot blot assay.

Once an antibody of interest of a combinatorial library has beenidentified and sufficiently characterized, the DNA sequences encodingthe light and heavy chains of said antibody are isolated by means ofstandardized molecular-biological techniques, for example, by means ofPCR amplification of DNA from the display package (e.g., the phage)which has been isolated during library screening. Nucleotide sequencesof genes for light and heavy antibody chains, which may be used forpreparing PCR primers, are known to one of ordinary skill in the art. Amultiplicity of such sequences are described, for example, in Kabat, E.A., et al. (1991) Sequences of Proteins of Immunological Interest, FifthEdition, U.S. Department of Health and Human Services, NIH PublicationNo. 91-3242 and in the database of sequences of the human germ lineVBASE.

An antibody or antibody moiety of the invention may be produced byrecombinantly expressing the genes for light and heavy immunoglobulinchains in a host cell. In order to recombinantly express an antibody, ahost cell is transfected with one or more recombinant expression vectorscarrying DNA fragments encoding the light and heavy immunoglobulinchains of said antibody, thereby expressing the light and heavy chainsin the host cell and secreting them preferably into the medium in whichsaid host cells are cultured. The antibodies can be isolated from thismedium. Standardized recombinant DNA methods are used in order to obtaingenes for heavy and light antibody chains, to insert said genes intorecombinant expression vectors and to introduce said vectors into hostcells. Methods of this kind are described, for example, in Sambrook,Fritsch and Maniatis (eds.), Molecular Cloning; A Laboratory Manual,Second Edition, Cold Spring Harbor, N.Y., (1989), Ausubel, F. M. et al.(eds.) Current Protocols in Molecular Biology, Greene PublishingAssociates, (1989) and in U.S. Pat. No. 4,816,397 by Boss et al.

Once DNA fragments encoding VH and VL segments of the antibody ofinterest have been obtained, said DNA fragments may be furthermanipulated using standardized recombinant DNA techniques, for example,in order to convert the genes for variable regions to genes for fulllength antibody chains, to genes for Fab fragments or to an scFv gene.These manipulations comprise linking a VL- or VH-encoding DNA fragmentoperatively to another DNA fragment encoding another protein, forexample a constant antibody region or a flexible linker. The term“operatively linked” is to be understood here as meaning that the twoDNA fragments are linked in such a way that the amino acid sequencesencoded by said two DNA fragments remain in frame. The isolated DNAencoding the VH region may be converted to a gene for a full lengthheavy chain by operatively linking the VH-region encoding DNA withanother DNA molecule encoding heavy chain constant regions (CH1, CH2 andCH3). The sequences of human heavy chain constant region genes are wellknown (see, for example, Kabat, E. A., et al. (1991) Sequences ofProteins of Immunological Interest, Fifth Edition, U.S. Department ofHealth and Human Services, NIH Publication No. 91-3242), and DNAfragments spanning said regions may be obtained by means of standardizedPCR amplification. The heavy chain constant region may be a constantregion from IgG1, IgG2, IgG3, IgG4, IgM, IgA, IgE or IgD, withpreference being given to a constant region from IgG, in particular IgG1or IgG4. To obtain a gene for a heavy chain Fab fragment, theVH-encoding DNA may be operatively linked to another DNA moleculeencoding merely the heavy chain constant region CH1. The isolated DNAencoding the VL region may be converted to a gene for a full lengthlight chain (and a gene for an Fab light chain) by operatively linkingthe VL-encoding DNA to another DNA molecule encoding the light chainconstant region CL. The sequences of genes of the constant region ofhuman light chain are well known (see Kabat, E. A., et al. (1991)Sequences of Proteins of Immunological Interest, Fifth Edition, U.S.Department of Health and Human Services, NIH Publication No. 91-3242),and DNA fragments spanning said regions may be obtained by means ofstandardized PCR amplification. The light chain constant region may be aconstant kappa or lambda region, a constant kappa region beingpreferred.

In order to generate an scFv gene, the VH- and VL-encoding DNA fragmentsmay be operatively linked to another fragment encoding a flexiblelinker, for example the amino acid sequence (Gly₄-Ser)₃ so that the VHand VL sequences are expressed as a continuous single-chain protein,with the VL and VH regions being linked to one another via said flexiblelinker (see Bird et al. (1988) Science 242:423-426; Huston et al. (1988)Proc. Natl. Acad. Sci. USA 85:5879-5883; McCafferty et al., Nature(1990) 348:552-554).

Single domain VH and VL having the binding affinities as described abovemay be isolated from single domain libraries by the above-describedmethods. Two VH single-domain chains (with or without CH1) or two VLchains or a pair of one VH chain and one VL chain with the desiredbinding affinity may be useful as described herein for the antibodies ofthe invention.

In order to express the recombinant antibodies or antibody moieties ofthe invention, the DNAs encoding partial or full length light and heavychains may be inserted into expression vectors so as to operatively linkthe genes to appropriate transcriptional and translational controlsequences. In this context, the term “operatively linked” is to beunderstood to mean that an antibody gene is ligated in a vector in sucha way that transcriptional and translational control sequences withinthe vector fulfill their intended function of regulating transcriptionand translation of said antibody gene. Expediently, the expressionvector and the expression control sequences are chosen so as to becompatible with the expression host cell used. The gene for the antibodylight chain and the gene for the antibody heavy chain may be insertedinto separate vectors or both genes are inserted into the sameexpression vector, this being the usual case. The antibody genes areinserted into the expression vector by means of standardized methods(for example by ligation of complementary restriction cleavage sites onthe antibody gene fragment and the vector, or by ligation of blunt ends,if no restriction cleavage sites are present). The expression vector mayalready carry sequences for antibody constant regions prior to insertionof the sequences for the light and heavy chains. For example, oneapproach is to convert the VH and VL sequences to full length antibodygenes by inserting them into expression vectors already encoding theheavy and, respectively, light chain constant regions, therebyoperatively linking the VH segment to the CH segment(s) within thevector and also operatively linking the VL segment to the CL segmentwithin the vector.

Additionally or alternatively, the recombinant expression vector mayencode a signal peptide which facilitates secretion of the antibodychain from the host cell. The gene for said antibody chain may be clonedinto the vector, thereby linking the signal peptide in frame to the Nterminus of the gene for the antibody chain. The signal peptide may bean immuno-globulin signal peptide or a heterologous signal peptide (i.e.a signal peptide from a non-immunoglobulin protein). In addition to thegenes for the antibody chain, the expression vectors of the inventionmay have regulatory sequences controlling expression of the genes forthe antibody chain in a host cell.

The term “regulatory sequence” is intended to include promoters,enhancers and further expression control elements (e.g. polyadenylationsignals) which control transcription or translation of the genes for theantibody chain. Regulatory sequences of this kind are described, forexample, in Goeddel; Gene Expression Technology: Methods in Enzymology185, Academic Press, San Diego, Calif. (1990). The skilled worker willappreciate that the expression vector design which includes selection ofregulatory sequences may depend on factors such as the choice of thehost cell to be transformed, the desired strength of expression of theprotein, etc. Preferred regulatory sequences for expression in mammalianhost cells include viral elements resulting in strong and constitutiveprotein expression in mammalian cells, such as promoters and/orenhancers derived from cytomegalovirus (CMV) (such as the CMVpromoter/enhancer), simian virus 40 (SV40) (such as the SV40promoter/enhancer), adenovirus (e.g., the adenovirus major late promoter(AdMLP)) and polyoma. For a further description of viral regulatoryelements and sequences thereof, see, for example, U.S. Pat. No.5,168,062 to Stinski, U.S. Pat. No. 4,510,245 to Bell et al. and U.S.Pat. No. 4,968,615 to Schaffner et al.

Apart from the genes for the antibody chain and the regulatorysequences, the recombinant expression vectors of the invention may haveadditional sequences such as those which regulate replication of thevector in host cells (e.g., origins of replication) and selectablemarker genes. The selectable marker genes facilitate the selection ofhost cells into which the vector has been introduced (see, for example,U.S. Pat. Nos. 4,399,216, 4,634,665 and 5,179,017, all to Axel et al.).For example, it is common for the selectable marker gene to render ahost cell into which the vector has been inserted resistant to cytotoxicdrugs such as G418, hygromycin or methotrexate. Preferred selectablemarker genes include the gene for dihydrofolate reductase (DHFR) (foruse in dhfr⁻ host cells with methotrexate selection/amplification) andthe neo gene (for G418 selection).

For expression of the light and heavy chains, the expression vector(s)encoding said heavy and light chains is(are) transfected into a hostcell by means of standardized techniques. The various forms of the term“transfection” are intended to comprise a multiplicity of techniquescustomarily used for introducing exogenous DNA into a prokaryotic oreukaryotic host cell, for example electroporation, calcium phosphateprecipitation, DEAE-dextran transfection, and the like. Although it istheoretically possible to express the antibodies of the invention eitherin prokaryotic or eukaryotic host cells, preference is given toexpressing the antibodies in eukaryotic cells and, in particular, inmammalian host cells, since the probability of a correctly folded andimmunologically active antibody being assembled and secreted is higherin such eukaryotic cells and in particular mammalian cells than inprokaryotic cells. Prokaryotic expression of antibody genes has beenreported as being ineffective for production of high yields of activeantibody (Boss, M. A. and Wood, C. R. (1985) Immunology Today 6:12-13).

Preferred mammalian host cells for expressing recombinant antibodies ofthe invention include CHO cells (including dhfr⁻ CHO cells described inUrlaub and Chasin, (1980) Proc. Natl. Acad. Sci. USA 77:4216-4220, whichare used together with a DHFR-selectable marker, as described, forexample, in R. J. Kaufman and P. A. Sharp (1982) Mol. Biol.159:601-621), NSO myeloma cells, COS cells and SP2 cells. Whenintroducing recombinant expression vectors encoding the antibody genesinto mammalian host cells, the antibodies are produced by culturing thehost cells until the antibody is expressed in said host cells or,preferably, the antibody is secreted into the culture medium in whichthe host cells grow. The antibodies may then be isolated from theculture medium by using standardized protein purification methods. It islikewise possible to use host cells in order to produce moieties ofintact antibodies, such as Fab fragments or scFv molecules. Variationsof the above-described procedure are of course included in theinvention. For example, it may be desirable to transfect a host cellwith DNA encoding either the light chain or the heavy chain (but notboth) of an antibody of the invention. If either light or heavy chainsare present which are not required for binding of the antigen ofinterest, then the DNA encoding either such a light or such a heavychain or both may be removed partially or completely by means ofrecombinant DNA technology. Molecules expressed by such truncated DNAmolecules are likewise included in the antibodies of the invention. Inaddition, it is possible to produce bifunctional antibodies in which aheavy chain and a light chain are an antibody of the invention and theother heavy chain and the other light chain have specificity for anantigen different from the antigen of interest, by crosslinking anantibody of the invention to a second antibody by means of standardizedchemical methods.

In a preferred system for recombinant expression of an antibody of theinvention or an antigen-binding moiety thereof, a recombinant expressionvector encoding both the antibody heavy chain and the antibody lightchain is introduced into dhfr⁻ CHO cells by means of calciumphosphate-mediated transfection. Within the recombinant expressionvector, the genes for the heavy and light antibody chains are in eachcase operatively linked to regulatory CMV enhancer/AdMLP-promoterelements in order to effect strong transcription of said genes. Therecombinant expression vector also carries a DHFR gene which can be usedfor selecting dhfr⁻ CHO cells transfected with the vector by usingmethotrexate selection/amplification. The selected transformed hostcells are cultured so that the heavy and light antibody chains areexpressed, and intact antibody is isolated from the culture medium.Standardized molecular-biological techniques are used in order toprepare the recombinant expression vector, to transfect the host cells,to select the transformants, to culture said host cells, and to obtainthe antibody from the culture medium. Thus, the invention relates to amethod of synthesizing a recombinant antibody of the invention byculturing a host cell of the invention in a suitable culture mediumuntil a recombinant antibody of the invention has been synthesized. Themethod may further comprise isolating said recombinant antibody fromsaid culture medium.

As an alternative to screening recombinant antibody libraries by phagedisplay, other methods known to the skilled worker may be used forscreening large combinatorial libraries to identify the antibodies ofthe invention. Basically, any expression system in which a closephysical linkage between a nucleic acid and the antibody encoded therebyis established and may be used to select a suitable nucleic acidsequence by virtue of the properties of the antibody it encodes may beemployed. In one type of an alternative expression system, therecombinant antibody library is expressed in the form of RNA-proteinfusions, as described in WO 98/31700 to Szostak and Roberts, and inRoberts, R. W. and Szostak, J. W. (1997) Proc. Natl. Acad. Sci. USA94:12297-12302. In this system, in-vitro translation of synthetic mRNAscarrying on their 3′ end puromycin, a peptidyl acceptor antibiotic,generates a covalent fusion of an mRNA and the peptide or proteinencoded by it. Thus, a specific mRNA of a complex mixture of mRNAs (e.g.a combinatorial library) may be concentrated on the basis of theproperties of the encoded peptide or protein (e.g. of the antibody or amoiety thereof), such as binding of said antibody or said moiety thereofto Aβ(12-42) globulomer or a derivative thereof. Nucleic acid sequenceswhich encode antibodies or moieties thereof and which are obtained byscreening of such libraries may be expressed by recombinant means in theabove-described manner (e.g. in mammalian host cells) and may, inaddition, be subjected to further affinity maturation by eitherscreening in further rounds mRNA-peptide fusions, introducing mutationsinto the originally selected sequence(s), or using other methods ofin-vitro affinity maturation of recombinant antibodies in theabove-described manner.

Combinations of In-Vivo and In-Vitro Approaches

The antibodies of the invention may likewise be produced by using acombination of in-vivo and in-vitro approaches such as methods in whichAβ(12-42) globulomer or a derivative thereof is first allowed to act onan antibody repertoire in a host animal in vivo to stimulate productionof Aβ(12-42) globulomer or derivative-binding antibodies and thenfurther antibody selection and/or antibody maturation (i.e.,optimization) are accomplished with the aid of one or more in-vitrotechniques. According to one embodiment, a combined method of this kindmay comprise firstly immunizing a nonhuman animal (e.g., a mouse, rat,rabbit, chicken, camelid, sheep or goat or a transgenic version thereofor a chimeric mouse) with said Aβ(12-42) globulomer or derivativethereof to stimulate an antibody response to the antigen and thenpreparing and screening a phage display antibody library by usingimmunoglobulin sequences of lymphocytes which have been stimulated invivo by the action of said Aβ(12-42) globulomer or derivative. The firststep of this combined procedure may be carried out in the mannerdescribed above in connection with the in-vivo approaches, while thesecond step of this procedure may be carried out in the manner describedabove in connection with the in-vitro approaches. Preferred methods ofhyperimmunizing nonhuman animals with subsequent in-vitro screening ofphage display libraries prepared from said stimulated lymphocytesinclude those described by BioSite Inc., see, for example, WO 98/47343,WO 91/17271, U.S. Pat. No. 5,427,908 and U.S. Pat. No. 5,580,717.

According to another embodiment, a combined method comprises firstlyimmunizing a nonhuman animal (e.g., a mouse, rat, rabbit, chicken,camelid, sheep, goat or a knockout and/or transgenic version thereof, ora chimeric mouse) with an Aβ(12-42) globulomer of the invention orderivative thereof to stimulate an antibody response to said Aβ(12-42)globulomer or derivative thereof and selecting the lymphocytes whichproduce the antibodies having the desired specificity by screeninghybridomas (prepared, for example, from the immunized animals). Thegenes for the antibodies or single domain antibodies are isolated fromthe selected clones (by means of standardized cloning methods such asreverse transcriptase polymerase chain reaction) and subjected toin-vitro affinity maturation in order to improve thereby the bindingproperties of the selected antibody or the selected antibodies. Thefirst step of this procedure may be conducted in the manner describedabove in connection with the in-vivo approaches, while the second stepof this procedure may be conducted in the manner described above inconnection with the in-vitro approaches, in particular by using methodsof in-vitro affinity maturation, such as those described in WO 97/29131and WO 00/56772.

In a further combined method, the recombinant antibodies are generatedfrom individual isolated lymphocytes by using a procedure which is knownto the skilled worker as selected lymphocyte antibody methods (SLAM) andwhich is described in U.S. Pat. No. 5,627,052, WO 92/02551 and Babcock,J. S. et al. (1996) Proc. Natl. Acad. Sci. USA 93:7843-7848. In thismethod, a nonhuman animal (e.g., a mouse, rat, rabbit, chicken, camelid,sheep, goat, or a transgenic version thereof, or a chimeric mouse) isfirstly immunized in vivo with Aβ(12-42) globulomer or a derivativethereof to stimulate an immune response to said oligomer or derivative,and then individual cells secreting antibodies of interest are selectedby using an antigen-specific haemolytic plaque assay. To this end, theglobulomer or derivative thereof or structurally related molecules ofinterest may be coupled to sheep erythrocytes, using a linker such asbiotin, thereby making it possible to identify individual cellssecreting antibodies with suitable specificity by using the haemolyticplaque assay. Following the identification of cells secreting antibodiesof interest, cDNAs for the variable regions of the light and heavychains are obtained from the cells by reverse transcriptase PCR, andsaid variable regions may then be expressed in association with suitableimmunoglobulin constant regions (e.g., human constant regions) inmammalian host cells such as COS or CHO cells. The host cellstransfected with the amplified immunoglobulin sequences derived from invivo-selected lymphocytes may then be subjected to further in-vitroanalysis and in-vitro selection by spreading out the transfected cells,for example, in order to isolate cells expressing antibodies with thebinding affinity. The amplified immunoglobulin sequences may furthermorebe manipulated in vitro.

Antibodies having the required affinities defined herein can be selectedby performing a dot blot essentially as described above. Briefly, theantigen is attached to a solid matrix, preferably dotted onto anitrocellulose membrane, in serial dilutions. The immobilized antigen isthen contacted with the antibody of interest followed by detection ofthe latter by means of an enzyme-conjugated secondary antibody and acolorimetric reaction; at defined antibody and antigen concentrations,the amount of antibody bound allows affinity determination. Thus therelative affinity of two different antibodies to one target, or of oneantibody to two different targets, is here defined as the relation ofthe respective amounts of target-bound antibody observed with the twoantibody-target combinations under otherwise identical dot blotconditions. Antibodies which bind to the same epitope as monoclonalantibody 10F4 or 3C5 can be obtained in a manner known per se.

In the same way as antibodies may be competing, described above,different target structures are herein said to be “competing” for aparticular antibody if at least one of these structures is capable ofspecifically reducing the measurable binding of another, preferably byoffering an overlapping or identical epitope, more preferably anidentical epitope. Competing target entities are useful for directlyselecting antibodies by virtue of their relative affinity to such targetstructures. Relative affinities may thus be determined directly by usinga competition assay in which distinguishable forms of the competingentities, e.g., differently labelled competing structures, are contactedwith the antibody of interest, and the relative affinity of the antibodyto each of these entities is deduced from the relative amounts of theseentities which are bound by the antibody. Such competition may be usedto directly enrich for antibodies possessing a desired relative affinityto the target entity, by attaching the entity towards which greateraffinity is desired to a solid matrix support and adding a suitableamount, preferably a molar excess, of the competing entity towards whichsmaller affinity is desired to the medium. Thus, the antibodiesdisplaying the desired relative affinities will tend to bind to thematrix more strongly than others and may be obtained after washing outthe less desirable forms, e.g., by washing out at low saltconcentrations and then harvesting the bound antibody by reversiblydetaching it from its target by using high salt concentrations. Ifdesired, several rounds of enrichment may be performed. In a particularembodiment of the invention, where the genotype underlying an antibodyis physically linked to this antibody, e.g., in a pool of hybridomas orantigen-displaying phages or yeast cells, the corresponding phenotypemay be rescued.

In another embodiment of the invention, a modified dot blot is usedwhere the immobilized antigen competes with a solved entity for antibodybinding, so that the relative affinity of the antibody can be deducedfrom the percentage bound to the immobilized antigen. Antibody moietiessuch as Fab and F(ab′)₂ fragments may be produced from whole antibodiesby using conventional techniques such as digestion with papain orpepsin. In addition, antibodies, antibody moieties and immunoadhesionmolecules may be obtained by using standardized recombinant DNAtechniques.

The present invention also relates to pharmaceutical agents(compositions) comprising an antibody of the invention and, optionally,a pharmaceutically suitable carrier. Pharmaceutical compositions of theinvention may furthermore contain at least one additional therapeuticagent, for example one or more additional therapeutic agents for thetreatment of a disease for whose relief the antibodies of the inventionare useful. If, for example, the antibody of the invention binds to aglobulomer of the invention, the pharmaceutical composition mayfurthermore contain one or more additional therapeutic agents useful forthe treatment of disorders in which the activity of said globulomer isimportant. Pharmaceutically suitable carriers include any solvents,dispersing media, coatings, antibacterial and antifungal agents,isotonic and absorption-delaying agents, and the like, as long as theyare physiologically compatible. Pharmaceutically acceptable carriersinclude, for example, water, saline, phosphate-buffered saline,dextrose, glycerol, ethanol and the like, and combinations thereof. Inmany cases, preference is given to using isotonic agents, for examplesugars, polyalcohols such as mannitol or sorbitol, or sodium chloride inaddition. Pharmaceutically suitable carriers may furthermore containrelatively small amounts of auxiliary substances such as wetting agentsor emulsifiers, preservatives or buffers, which increase the half lifeor efficacy of the antibodies. The pharmaceutical compositions may besuitable for parenteral administration, for example. Here, theantibodies are prepared preferably as injectable solutions with anantibody content of 0.1-250 mg/mL. The injectable solutions may beprepared in liquid or lyophilized form, the dosage form being a flintglass or vial, an ampoule or a filled syringe. The buffer may containL-histidine (1-50 mM, preferably 5-10 mM) and have a pH of 5.0-7.0,preferably of 6.0. Further suitable buffers include, without beinglimited thereto, sodium succinate, sodium citrate, sodium phosphate orpotassium phosphate buffers. Sodium chloride may be used in order toadjust the tonicity of the solution to a concentration of 0-300 mM(preferably 150 mM for a liquid dosage form). Cryoprotectants, forexample sucrose (e.g., 0-100, preferably 0.5-1.00) may also be includedfor a lyophilized dosage form. Other suitable cryoprotectants aretrehalose and lactose. Fillers, for example mannitol (e.g., 1-100,preferably 2-40) may also be included for a lyophilized dosage form.Stabilizers, for example L-methionine (e.g., 51-50 mM, preferably 5-10mM) may be used both in liquid and lyophilized dosage forms. Furthersuitable fillers are glycine and arginine. Surfactants, for example,polysorbate 80 (e.g., 0-0.050, preferably 0.005-0.010), may also beused. Further surfactants are polysorbate 20 and BRIJ surfactants.

The compositions of the invention may have a multiplicity of forms.These include liquid, semisolid and solid dosage forms, such as liquidsolutions (e.g., injectable and infusible solutions), dispersions orsuspensions, tablets, pills, powders, liposomes and suppositories. Thepreferred form depends on the intended type of administration and on thetherapeutic application. Typically, preference is given to compositionsin the form of injectable or infusible solutions, for examplecompositions which are similar to other antibodies for passiveimmunization of humans. The preferred route of administration isparenteral (e.g., intravenous, subcutaneous, intraperitoneal orintramuscular). According to a preferred embodiment, the antibody isadministered by intravenous infusion or injection. According to anotherpreferred embodiment, the antibody is administered by intramuscular orsubcutaneous injection. Therapeutic compositions must typically besterile and stable under preparation and storage conditions. Thecompositions may be formulated as solutions, microemulsions,dispersions, liposomes or other ordered structures suitable for highconcentrations of active substance. Sterile injectable solutions may beprepared by introducing the active compound (i.e., the antibody) in therequired amount into a suitable solvent, where appropriate with one or acombination of the abovementioned ingredients, as required, and thensterile-filtering said solution. Dispersions are usually prepared byintroducing the active compound into a sterile vehicle containing abasic dispersion medium and, where appropriate, other requiredingredients. In the case of a sterile lyophilized powder for preparingsterile injectable solutions, vacuum drying and spray drying arepreferred methods of preparation, which produces a powder of the activeingredient and, where appropriate, of further desired ingredients from apreviously sterile-filtered solution. The correct flowability of asolution may be maintained by using, for example, a coating such aslecithin, by maintaining, in the case of dispersions the requiredparticle size or by using surfactants. A prolonged absorption ofinjectable compositions may be achieved by additionally introducing intothe composition an agent which delays absorption, for examplemonostearate salts and gelatine.

The antibodies of the invention may be administered by a multiplicity ofmethods known to the skilled worker, although the preferred type ofadministration for many therapeutic applications is subcutaneousinjection, intravenous injection or infusion. The skilled worker willappreciate that the route and/or type of administration depend on theresult desired. According to particular embodiments, the active compoundmay be prepared with a carrier which protects the compound against rapidrelease, such as, for example, a formulation with sustained orcontrolled release, which includes implants, transdermal plasters andmicroencapsulated release systems. Biologically degradable biocompatiblepolymers such as ethylene vinyl acetate, polyanhydrides, polyglycolicacid, collagen, polyorthoesters and polylactic acid may be used. Themethods of preparing such formulations are well known to the skilledworker; see, for example, Sustained and Controlled Release Drug DeliverySystems, J. R. Robinson, ed., Marcel Dekker, Inc., New York, 1978.

According to particular embodiments, an antibody of the invention may beadministered orally, for example, in an inert diluent or a metabolizableedible carrier. The antibody (and further ingredients, if desired) mayalso be enclosed in a hard or soft gelatine capsule, compressed totablets or added directly to food. For oral therapeutic administration,the antibodies may be mixed with excipients and used in the form of oraltablets, buccal tablets, capsules, elixirs, suspensions, syrups and thelike. If it is intended to administer an antibody of the invention via aroute other than the parenteral one, it may be necessary to choose acoating from a material which prevents its inactivation.

The present invention also relates to a method of inhibiting theactivity of globulomers of the invention in an individual which suffersfrom a disorder in which the amyloid β protein is involved and in whichin particular the activity of said globulomers of the invention isimportant. Said method comprises the administration of at least oneantibody of the invention to the individual with the aim of inhibitingthe activity of the globulomer to which the antibody binds. Saidindividual is preferably a human being. An antibody of the invention maybe administered for therapeutic purposes to a human individual. Inaddition, an antibody of the invention may be administered to a nonhumanmammal for veterinary purposes or within the framework of an animalmodel for a particular disorder. Such animal models may be useful forevaluating the therapeutic efficacy of antibodies of the invention (forexample for testing dosages and time courses of administration).

Disorders in which the globulomers of the invention play a part include,in particular, disorders in whose development and/or progression aglobulomer of the invention is involved. These are in particular thosedisorders in which globulomers of the invention are evidently orpresumably responsible for the pathophysiology of said disorder or are afactor which contributes to the development and/or progression of saiddisorder. Accordingly, those disorders are included here in whichinhibition of the activity of globulomers of the invention can relievesymptoms and/or progression of the disorder. Such disorders can beverified, for example, by an increased concentration of globulomers ofthe invention in a biological fluid of an individual suffering from aparticular disorder (e.g., increased concentration in serum, plasma,CSF, urine, etc.). This may be detected, for example, by using anantibody of the invention. The globulomers of the invention play animportant part in the pathology associated with a multiplicity ofdisorders in which neurodegenerative elements, cognitive deficiencies,neurotoxic elements and inflammatory elements are involved.

In another aspect of the invention, disorders that can be treated orprevented include those associated with amyloidoses. The term“amyloidoses” herein denotes a number of disorders characterized byabnormal folding, clumping, aggregation and/or accumulation ofparticular proteins (amyloids, fibrous proteins and their precursors) invarious tissues of the body. In Alzheimer's disease and Down's syndrome,nerve tissue is affected, and in cerebral amyloid angiopathy (CAA) bloodvessels are affected.

The pharmaceutical compositions of the invention may include a“therapeutically effective amount” or a “prophylactically effectiveamount” of an antibody or antibody moiety of the invention. A“therapeutically effective amount” refers to an amount effective, atdosages and for periods of time necessary, to achieve the desiredtherapeutic result. A therapeutically effective amount of the antibodyor antibody moiety may be determined by a person skilled in the art andmay vary according to factors such as the disease state, age, sex, andweight of the individual, and the ability of the antibody or antibodymoiety to elicit a desired response in the individual. A therapeuticallyeffective amount is also one in which any toxic or detrimental effectsof the antibody or antibody portion are outweighed by thetherapeutically beneficial effects. A “prophylactically effectiveamount” refers to an amount effective, at dosages and for periods oftime necessary, to achieve the desired prophylactic result. Typically,since a prophylactic dose is used in subjects prior to or at an earlierstage of disease, the prophylactically effective amount will be lessthan the therapeutically effective amount.

Moreover, the present invention includes a further method of preventingor treating Alzheimer's disease in a patient in need of such preventionor treatment. This method comprises the step of administering thevaccine noted above to the patient in an amount sufficient to effect theprevention or treatment.

Further, the present invention encompasses a method of identifyingcompounds suitable for active immunization of a patient predicted todevelop an amyloidosis, e.g. Alzheimer's disease. This methodcomprises: 1) exposing one or more compounds of interest to one or moreof the antibodies described above for a time and under conditionssufficient for the one or more compounds to bind to the antibody orantibodies; 2) identifying those compounds which bind to the antibody orantibodies, the identified compounds to be used in active immunizationin a patient predicated to develop an amyloidosis, e.g., Alzheimer'sdisease.

Within the framework of diagnostic usage of the antibodies, qualitativeor quantitative specific globulomer determination serves in particularto diagnose disease-relevant amyloid β forms. In this context,specificity means the possibility of being able to detect a particularglobulomer or a derivative thereof, or a mixture thereof with sufficientsensitivity. The antibodies of the invention advantageously havedetection threshold concentrations of less than 10 ng/mL of sample,preferably of less than 1 ng/mL of sample and particularly preferably ofless than 100 pg/mL of sample, meaning that at least the concentrationof globulomer per mL of sample, indicated in each case, advantageouslyalso lower concentrations, can be detected by the antibodies of theinvention. The detection is carried out immunologically. This may becarried out, in principle, by using any analytical or diagnostic assaymethod in which antibodies are used, including agglutination andprecipitation techniques, immunoassays, immunohistochemical methods andimmunoblot techniques, for example Western blotting or, preferably, dotblot methods. In vivo methods, for example imaging methods, are alsoincluded here.

The use in immunoassays is advantageous. Competitive immunoassays, i.e.,assays where antigen and labelled antigen (tracer) compete for antibodybinding, and sandwich immunoassays, i.e., assays where binding ofspecific antibodies to the antigen is detected by a second, usuallylabelled antibody, are both suitable. These assays may be eitherhomogeneous, i.e., without separation into solid and liquid phases, orheterogeneous, i.e., bound labels are separated from unbound ones, forexample, via solid phase-bound antibodies. Depending on labelling andmethod of measurement, the various heterogeneous and homogeneousimmunoassay formats can be classified into particular classes, forexample RIAs (radioimmunoassays), ELISA (enzyme-linked immunosorbentassay), FIA (fluorescence immunoassay), LIA (luminescence immunoassay),TRFIA (time-resolved FIA), IMAC (immunoactivation), EMIT(enzyme-multiplied immune test), TIA (turbidometric immunoassay), I-PCR(immuno-PCR).

For the globulomer quantification of the invention, preference is givento competitive immunoassays in which a defined amount of labelledglobulomer derivative serving as tracer competes with the globulomer ofthe sample (containing an unknown amount of unlabelled globulomers) tobe quantified for binding to the antibody used. The amount of antigen,i.e., the amount of globulomer, in the sample can be determined from theamount of the displaced tracer with the aid of a standard curve.

Of the labels available for these purposes, enzymes have provedadvantageous. Systems based on peroxidases, in particular, horseradishperoxidase, alkaline phosphatase and β-D-galactosidase, may be used, forexample. Specific substrates whose conversion can be monitoredphotometrically, for example, are available for these enzymes. Suitablesubstrate systems are based on p-nitrophenyl phosphate (p-NPP),5-bromo-4-chloro-3-indolyl phosphate/nitroblue tetrazolium (BCIP/NPT),Fast-Red/naphthol-AS-TS phosphate for alkaline phosphatase;2,2-azinobis(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS),o-phenylenediamine (OPT), 3,3′,5,5′-tetramethylbenzidine (TMB),o-dianisidine, 5-aminosalicylic acid, 3-dimethylaminobenzoic acid (DMAB)and 3-methyl-2-benzothiazolinehydrazone (MBTH) for peroxidases;o-nitrophenyl-β-D-galactoside (o-NPG), p-nitrophenyl-β-D-galactoside and4-methylumbelliphenyl-β-D-galactoside (MUG) for β-D-galactosidase. Inmany cases, these substrate systems are commercially available in aready-to-use form, for example in the form of tablets which may alsocontain further reagents such as appropriate buffers and the like. Thetracers used may be labelled globulomers. In this sense, a particularglobulomer can be determined by labelling the globulomer to bedetermined and using it as tracer. The coupling of labels to globulomersfor preparing tracers may be carried out in a manner known per se. Thecomments above on derivatization of globulomers of the invention arereferred to by analogy. In addition, a number of labels appropriatelymodified for conjugation to proteins are available, for example biotin-,avidin-, extravidin- or streptavidin-conjugated enzymes,maleimide-activated enzymes and the like. These labels may be reacteddirectly with the oligomer or, if required, with the appropriatelyderivatized globulomer to give the tracer. If, for example, astreptavidin-peroxidase conjugate is used, then this firstly requiresbiotinylation of the globulomer. This applies correspondingly to thereverse order. Suitable methods to this end are also known to theskilled worker.

If a heterogeneous immunoassay format is chosen, the antigen-antibodycomplex may be separated by binding it to the support, for example viaan anti-idiotypical antibody coupled to said support, e.g. an antibodydirected against rabbit IgG. Appropriate supports, in particularmicrotiter plates coated with appropriate antibodies, are known andpartly commercially available.

The present invention further relates to immunoassay sets having atleast one antibody as described above and further components. Said setsare, usually in the form of a packaging unit, a combination of means forcarrying out a globulomer determination of the invention. For thepurpose of as easy handling as possible, said means are preferablyprovided in an essentially ready-to-use form. An advantageousarrangement offers the immunoassay in the form of a kit. A kit usuallycomprises multiple containers for separate arrangement of components.All components may be provided in a ready-to-use dilution, as aconcentrate for diluting or as a dry substance or lyophilisate fordissolving or suspending; individual or all components may be frozen orstored at room temperature until use. Sera are preferably shock-frozen,for example at −20° C. so that in these cases an immunoassay has to bekept preferably at temperatures below freezing prior to use. Furthercomponents supplied with the immunoassay depend on the type of saidimmunoassay. Usually, standard protein, tracer which may or may not berequired and control serum are supplied together with the antiserum.Furthermore, microtiter plates, preferably antibody-coated, buffers, forexample, for testing, for washing or for conversion of the substrate,and the enzyme substrate itself may also be included.

General principles of immunoassays and generation and use of antibodiesas auxiliaries in laboratory and hospital can be found, for example, inAntibodies, A Laboratory Manual (Harlow, E., and Lane, D., Ed., ColdSpring Harbor Laboratory, Cold Spring Harbor, N.Y., 1988).

The present invention also includes a method of diagnosing anamyloidosis, e.g., Alzheimer's disease, in a patient suspected of havingthis disease. This method comprises the steps of: 1) isolating abiological sample from the patient; 2) contacting the biological samplewith at least one of the antibodies described above for a time and underconditions sufficient for formation of antigen/antibody complexes; and3) detecting presence of the antigen/antibody complexes in said sample,presence of the complexes indicating a diagnosis of an amyloidosis,e.g., Alzheimer's disease, in the patient. The antigen may be, forexample, an globulomer or a portion or fragment thereof which has thesame functional properties as the full globulomer (e.g., bindingactivity).

Further, the present invention includes another method of diagnosing anamyloidosis, e.g., Alzheimer's disease in a patient suspected of havingthis disease. This method comprising the steps of: 1) isolating abiological sample from the patient; 2) contacting the biological samplewith an antigen for a time and under conditions sufficient for theformation of antibody/antigen complexes; 3) adding a conjugate to theresulting antibody/antigen complexes for a time and under conditionssufficient to allow the conjugate to bind to the bound antibody, whereinthe conjugate comprises one of the antibodies described above, attachedto a signal generating compound capable of generating a detectablesignal; and 4) detecting the presence of an antibody which may bepresent in the biological sample, by detecting a signal generated by thesignal generating compound, the signal indicating a diagnosis of anamyloidosis, e.g., Alzheimer's disease in the patient. The antigen maybe a globulomer or a portion or fragment thereof having the samefunctional properties as the full globulomer (e.g., binding activity).

The present invention includes an additional method of diagnosing anamyloidosis, e.g., Alzheimer's disease, in a patient suspected of havingan amyloidosis, e.g., Alzheimer's disease. This method comprises thesteps of: 1) isolating a biological sample from said patient; 2)contacting the biological sample with anti-antibody, wherein theanti-antibody is specific for one of the antibodies described above, fora time and under conditions sufficient to allow for formation ofanti-antibody/antibody complexes, the complexes containing antibodypresent in the biological sample; 2) adding a conjugate to resultinganti-antibody/antibody complexes for a time and under conditionssufficient to allow the conjugate to bind to bound antibody, wherein theconjugate comprises an antigen, which binds to a signal generatingcompound capable of generating a detectable signal; and 3) detecting asignal generated by the signal generating compound, the signalindicating a diagnosis of an amyloidosis, e.g., Alzheimer's disease, inthe patient.

Also, the present invention includes a kit comprising: a) at least oneof the antibodies described above and b) a conjugate comprising anantibody attached to a signal-generating compound, wherein the antibodyof the conjugate is different from the isolated antibody.

The present invention also encompasses a kit comprising: a) ananti-antibody to one of the antibodies described above and b) aconjugate comprising an antigen attached to a signal-generatingcompound. The antigen may be a globulomer or a fragment or portionthereof having the same functional characteristics as the globulomer(e.g., binding activity).

In one diagnostic embodiment of the present invention, an antibody ofthe present invention, or a portion thereof, is coated on a solid phase(or is present in a liquid phase). The test or biological sample (e.g.,whole blood, cerebrospinal fluid, serum, etc.) is then contacted withthe solid phase. If antigen (e.g., globulomer) is present in the sample,such antigens bind to the antibodies on the solid phase and are thendetected by either a direct or indirect method. The direct methodcomprises simply detecting presence of the complex itself and thuspresence of the antigens. In the indirect method, a conjugate is addedto the bound antigen. The conjugate comprises a second antibody, whichbinds to the bound antigen, attached to a signal-generating compound orlabel. Should the second antibody bind to the bound antigen, thesignal-generating compound generates a measurable signal. Such signalthen indicates presence of the antigen in the test sample. Examples ofsolid phases used in diagnostic immunoassays are porous and non-porousmaterials, latex particles, magnetic particles, microparticles (see U.S.Pat. No. 5,705,330), beads, membranes, microtiter wells and plastictubes. The choice of solid phase material and method of labeling theantigen or antibody present in the conjugate, if desired, are determinedbased upon desired assay format performance characteristics.

As noted above, the conjugate (or indicator reagent) will comprise anantibody (or perhaps anti-antibody, depending upon the assay), attachedto a signal-generating compound or label. This signal-generatingcompound or “label” is itself detectable or may be reacted with one ormore additional compounds to generate a detectable product. Examples ofsignal-generating compounds include chromogens, radioisotopes (e.g.,125I, 131I, 32P, 3H, 35S and 14C), chemiluminescent compounds (e.g.,acridinium), particles (visible or fluorescent), nucleic acids,complexing agents, or catalysts such as enzymes (e.g., alkalinephosphatase, acid phosphatase, horseradish peroxidase,beta-galactosidase and ribonuclease). In the case of enzyme use (e.g.,alkaline phosphatase or horseradish peroxidase), addition of a chromo-,fluoro-, or lumo-genic substrate results in generation of a detectablesignal. Other detection systems such as time-resolved fluorescence,internal-reflection fluorescence, amplification (e.g., polymerase chainreaction) and Raman spectroscopy are also useful. Examples of biologicalfluids which may be tested by the above immunoassays include plasma,whole blood, dried whole blood, serum, cerebrospinal fluid or aqueous ororgano-aqueous extracts of tissues and cells.

The present invention also encompasses a method for detecting thepresence of antibodies in a test sample. This method comprises the stepsof: (a) contacting the test sample suspected of containing antibodieswith anti-antibody specific for the antibodies in the patient sampleunder time and conditions sufficient to allow the formation ofanti-antibody/antibody complexes, wherein the anti-antibody is anantibody of the present invention which binds to an antibody in thepatient sample; (b) adding a conjugate to the resultinganti-antibody/antibody complexes, the conjugate comprising an antigen(which binds to the anti-antibody) attached to a signal generatingcompound capable of detecting a detectable signal; and (d) detecting thepresence of the antibodies which may be present in the test sample bydetecting the signal generated by the signal generating compound. Acontrol or calibrator may be used which comprises antibody to theanti-antibody.

Kits are also included within the scope of the present invention. Morespecifically, the present invention includes kits for determining thepresence of antigens (e.g., globulomers) in a patient suspected ofhaving Alzheimer's disease or another condition characterized bycognitive impairment. In particular, a kit for determining the presenceof antigens in a test sample comprises a) an antibody as defined hereinor moiety thereof; and b) a conjugate comprising a second antibody(having specificity for the antigen) attached to a signal generatingcompound capable of generating a detectable signal. The kit may alsocontain a control or calibrator which comprises a reagent which binds tothe antigen as well as a package insert describing the procedure to beused when conducting the assay.

The present invention also includes a kit for detecting antibodies in atest sample. The kit may comprise a) an anti-antibody specific (forexample, one of the subject invention) for the antibody of interest, andb) an antigen or portion thereof as defined above. A control orcalibrator comprising a reagent which binds to the antigen may also beincluded. More specifically, the kit may comprise a) an anti-antibody(such as the one of the present invention) specific for the antibody andb) a conjugate comprising an antigen (e.g., globulomer) attached to asignal generating compound capable of generating a detectable signal.Again, the kit may also comprise a control of calibrator comprising areagent which binds to the antigen as well as a package insertdescribing the components of the kits and how they are to be utilized.

The kit may also comprise one container such as vial, bottles or strip,with each container with a pre-set solid phase, and other containerscontaining the respective conjugates. These kits may also contain vialsor containers of other reagents needed for performing the assay, such aswashing, processing and indicator reagents.

It should also be noted that the subject invention not only includes thefull length antibodies described above but also moities or fragmentsthereof, for example, the Fab portion thereof. Additionally, the subjectinvention encompasses any antibody having the same properties of thepresent antibodies in terms of, for example, binding specificity,structure, etc.

ADVANTAGES OF THE INVENTION

By immunization with Aβ(12-42) globulomer (as described in Example I),different monoclonal antibodies may be obtained which differ in theirtolerance or recognition of different Aβ(1-42) oligomers and Aβ(X-42)oligomers, as determined by comparative dot blotting as described above.This allows development of an antibody directed to Aβ oligomers whichpossesses an optimal relation between cognition enhancing effect,desired specificity over other Aβ forms and minimal side effect profile.The same holds true for monoclonal antibodies for use in passiveimmunization. The advantage of such a specific strategy for immunization(active and passive) is that it will not induce an immune responseagainst Aβ monomers, Aβ peptides in fibrillary states of aggregation orsAPPα. This is advantageous in several ways:

-   -   1) In the form of insoluble Aβ plaques, Aβ peptides in        fibrillary states of aggregation amount to the major part of the        entire Aβ peptide pool in AD brains. A massive release of Aβ by        dissolution of Aβ plaques induced by reaction of anti-Aβ        antibodies with these plaques is to be regarded as detrimental.        This massive release of Aβ would then cross the blood-brain        barrier, enter the bloodstream and potentially increase the risk        of microhaemorrhages. In addition, in the ELAN trial mentioned        above, this very strategy of immunization with fibrillary Aβ        peptide forms required cancellation of the trial due to 60 of        cases with an onset of meningoencephalitis.    -   2) Immune responses directed to monomeric Aβ peptide forms are        undesirable, as it could be shown that the latter may exert        cognition-enhancing effects.    -   3) Immune responses directed to sAPPα are likewise undesirable,        as this might lead to a reaction with the physiologically        occurring precursor protein APP and thus to an autoimmune        reaction. Moreover, sAPPα was also shown to exert        cognition-enhancing effects.

4) A response directed to vascular Aβ peptide in the form of CAA is tobe avoided in order to eschew the undesirable side effect ofmicrohaemorrhages (i.e., antibodies against the central portion of Aβand which in addition do not bind to Aβ-peptides aggregated in the formof CAA induce fewer microhaemorrhages when compared to such against theN-terminus, see above).

-   -   5) Antibodies which specifically react with Aβ oligomers will        have higher bioavailability with regard to the        pathophysiologically relevant Aβ species, as they will not be        bound to, e.g., fibrillary or monomeric Aβ and thus made        unavailable for therapeutic effect.        Again, it should be noted that the antibodies of the present        invention and, in particular, 10F4 and 3C5, do not (or with a        lower binding affinity compared to commercially available        antibodies like 6E10 (Signet Cat. no.: 9320)) detect amyloid        beta in the cerebrospinal fluid. Thus, due to the high turnover        rates of amyloid beta in the CSF, this lack of binding by the        antibodies to the amyloid beta in the CSF prevents the waste of        antibodies, as well as creates a more efficacious and selective        system in comparison to those antibodies which bind to all        amyloid beta found in the body (e.g., brain and CSF). Further,        this property of the antibodies of the present invention allows        one to reduce the amount of antibody to be administered (in        connection with passive immunization), reduces the risk of side        effects since the dose is lower thereby restricting antibodies        to the target, increases efficacy, and also increases the        therapeutic index. Furthermore, the risk of microhemmorhages is        also reduced. Additionally, since the antibodies do not detect        fibrillar forms of amyloid beta, the risks associated with such        complex formation are also reduced.

DEPOSIT INFORMATION

The hybridoma (ML45-3C5.5C10) which produces monoclonal antibody 3C5 wasdeposited with the American Type Culture Collection, 10801 UniversityBoulevard, Manassas, Va. 20110 on Feb. 28, 2006 under the terms of theBudapest Treaty and was assigned ATCC No. PTA-7406. Hybridoma(ML43-10F4.3H8) which produces monoclonal antibody 10F4 was depositedwith the American Type Culture Collection, 10801 University Boulevard,Manassas, Va. 20110 on Aug. 16, 2006 under the terms of the BudapestTreaty and was assigned ATCC No. PTA-7808.

The present invention may be illustrated by use of the followingnon-limiting examples:

Example I Preparation of Aβ(12-42) Globulomer for Immunization

The Aβ(12-42) synthetic peptide (AnaSpec Inc.; Lot #40443) was suspendedin 1000 (v/v) 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) at 40 mg/mL (5 mgin 125 μL HFIP) and incubated for complete solubilization under shakingat 37° C. for 1 h. The HFIP acts as a hydrogen-bond breaker and is usedto eliminate pre-existing structural inhomogeneities in the Aβ peptide.After centrifugation at 10000 g for 10 min the supernatant of theHFIP-dissolved Aβ(12-42) was diluted with 6.1 mL phosphate-bufferedsaline (PBS) (20 mM NaH₂PO₄, 140 mM NaCl, pH 7.4) and 625 μL 2% (w/v)sodium dodecyl sulfate (SDS) (in H₂O) were added (final concentration of0.2% (w/v) SDS) and incubated for 3 h at 37° C. Once more, 625 μL 2%(w/v) sodium dodecyl sulfate (SDS) (in H₂O) were added (finalconcentration of 0.4% (w/v) SDS) and further incubated for 3 h at 37° C.The solution was diluted with 7 mL H₂O and incubated for 16 h at 37° C.After centrifugation at 3000 g for 10 min the supernatant was furtherdiluted with 15 mL PBS (20 mM NaH₂PO₄, 140 mM NaCl, pH 7.4) andconcentrated by ultrafiltration (5 kDa cut-off) to 0.65 mL, dialysedagainst 20 mM NaH₂PO₄, 140 mM NaCl, 0.05% (w/v) SDS, pH 7.4 for 16 h atroom temperature, centrifuged at 10000 g for 10 min and the supernatantcomprising the Aβ(12-42) globulomer withdrawn. The samples werealiquoted and stored at −80° C. until further use.

Example II Production of Monoclonal Antibodies 3C5 AND 10F4

Balb/c mice were immunized sub-cutaneous with 50 μg of Aβ (12-42)globulomer as described in Example I in CFA (Sigma) and boosted twice atone month intervals. Spleens were collected and spleen cells fused withmouse myeloma SP2/0 cells at 5:1 ratio by a PEG procedure. Fusion cellswere plated in 96-well dishes in Azaserine/Hypoxanthine selection mediaat 2×10⁵ cells/mL, 200 mL per well. Cells were allowed to grow to formvisible colonies and supernatants assayed for Aβ oligomer reactivity bya direct ELISA assay. Hybridomas secreting antibodies to Aβ oligomerswere subcloned by limiting dilution, until antibody expression appearedstable.

Example III Dot-Blot Profile of the Selectivity of the Anti-AβGlobulomer Antibodies

In order to characterize the selectivity of the monoclonal anti-Aβglobulomer antibodies, they were probed for recognition with differentAβ-forms. To this end, serial dilutions of the individual Aβ formsranging from 100 pmol/μL to 0.01 pmol/μL in PBS supplemented with 0.2mg/mL BSA were made. 1 μL of each sample was blotted onto anitrocellulose membrane. For detection, the corresponding antibody wasused (0.2 μg/mL). Immunostaining was done using peroxidase conjugatedanti-mouse-IgG and the staining reagent BM Blue POD Substrate (Roche).

Aβ-Standards for Dot-Blot:

1. Aβ(1-42) monomer, 0.1% NH₄OH

-   -   1 mg Aβ(1-42) (Bachem Inc., Cat. no.: H-1368) were dissolved in        0.5 mL 0.1% NH₄OH in H₂O (freshly prepared) (=2 mg/mL) and        immediately shaken for 30 sec at room temperature to obtain a        clear solution. The sample was stored at −20° C. for further        use.

2. Aβ(1-40) Monomer, 0.1% NH₄OH

-   -   1 mg Aβ(1-40) (Bachem Inc., cat. no. H-1368) were dissolved in        0.5 mL 0.1% NH₄OH in H₂O (freshly prepared) (=2 mg/mL) and        immediately shaken for 30 sec. at room temperature to obtain a        clear solution. The sample was stored at −20° C. for further        use.

3. Aβ(1-42) monomer, 0.1% NaOH

-   -   2.5 mg Aβ(1-42) (Bachem Inc., cat. no. H-1368) were dissolved in        0.5 mL 0.1% NaOH in H₂O (freshly prepared) (=5 mg/mL) and        immediately shaken for 30 sec. at room temperature to obtain a        clear solution. The sample was stored at −20° C. for further        use.

4. Aβ(1-40) monomer, 0.1% NaOH

-   -   2.5 mg Aβ(1-40) (Bachem Inc., cat. no. H-1368) were dissolved in        0.5 mL 0.1% NaOH in H₂O (freshly prepared) (=5 mg/mL) and        immediately shaken for 30 sec. at room temperature to obtain a        clear solution. The sample was stored at −20° C. for further        use.

5. Aβ(1-42) Globulomer

-   -   The Aβ(1-42) synthetic peptide (H-1368, Bachem, Bubendorf,        Switzerland) was suspended in 100%        1,1,1,3,3,3-hexafluoro-2-propanol (HFIP) at 6 mg/mL and        incubated for complete solubilization under shaking at 37° C.        for 1.5 h. The HFIP acts as a hydrogen-bond breaker and is used        to eliminate pre-existing structural inhomogeneities in the Aβ        peptide. HFIP was removed by evaporation in a SpeedVac and        Aβ(1-42) resuspended at a concentration of 5 mM in        dimethylsulfoxide and sonicated for 20 s. The HFIP-pre-treated        Aβ(1-42) was diluted in phosphate-buffered saline (PBS) (20 mM        NaH₂PO₄, 140 mM NaCl, pH 7.4) to 400 μM and 1/10 volume 2%        sodium dodecyl sulfate (SDS) (in H₂O) added (final concentration        of 0.2% SDS). An incubation for 6 h at 37° C. resulted in the        16/20-kDa Aβ(1-42) globulomer (short form for globular oligomer)        intermediate. The 38/48-kDa Aβ(1-42) globulomer was generated by        a further dilution with three volumes of H₂O and incubation for        18 h at 37° C. After centrifugation at 3000 g for 20 min the        sample was concentrated by ultrafiltration (30-kDa cut-off),        dialysed against 5 mM NaH₂PO₄, 35 mM NaCl, pH 7.4, centrifuged        at 10000 g for 10 min and the supernatant comprising the        38/48-kDa Aβ(1-42) globulomer withdrawn. As an alternative to        dialysis the 38/48-kDa Aβ(1-42) globulomer can also be        precipitated by a ninefold excess (v/v) of ice-cold        methanol/acetic acid solution (33% methanol, 4% acetic acid) for        1 h at 4° C. The 38/48-kDa Aβ(1-42) globulomer is then pelleted        (10 min at 16200 g), resuspended in 5 mM NaH₂PO₄, 35 mM NaCl, pH        7.4, and the pH adjusted to 7.4.

6. Aβ(12-42) Globulomer

-   -   2 mL of an Aβ(1-42) globulomer preparation prepared according to        Example 3.5 (see above) are admixed with 38 mL buffer (5 mM        sodium phosphate, 35 mM sodium chloride, pH 7.4) and 150 μl of a        1 mg/mL GluC endoproteinase (Roche) in H₂O. The reaction mixture        is stirred for 6 h at RT, and a further 150 μl of a 1 mg/mL GluC        endoproteinase (Roche) in H₂O are subsequently added. The        reaction mixture is stirred at RT for another 16 h, followed by        addition of 8 μL of a 5 M DIFP (Diisopropylfluorphosphate)        solution. The reaction mixture is concentrated to approx. 1 mL        via a 15 mL 30 kDa Centriprep tube. The concentrate is admixed        with 9 mL of buffer (5 mM sodium phosphate, 35 mM sodium        chloride, pH 7.4) and again concentrated to 1 mL. The        concentrate is dialyzed at 6° C. against 1 L of buffer (5 mM        sodium phosphate, 35 mM NaCl) in a dialysis tube for 16 h. The        dialysate is adjusted to an SDS content of 0.1% with a 1%        strength SDS solution in H₂O. The sample is removed by        centrifugation at 10000 g for 10 min and the supernatant is        removed.

7. Aβ(20-42) Globulomer

-   -   1.59 mL of Aβ(1-42) globulomer preparation prepared according to        Example 2.5 (see above) are admixed with 38 mL of buffer (50 mM        MES/NaOH, pH 7.4) and 200 μL of a 1 mg/mL thermolysin solution        (Roche) in H₂O. The reaction mixture is stirred at RT for 20 h.        Then 80 μl of a 100 mM EDTA solution, pH 7.4, in H₂O are added        and the mixture is furthermore adjusted to an SDS content of        0.01% with 400 μl of a 1% strength SDS solution. The reaction        mixture is concentrated to approx. 1 mL via a 15 mL 30 kDa        Centriprep tube. The concentrate is admixed with 9 mL of buffer        (50 mM MES/NaOH, 0.02% SDS, pH 7.4) and again concentrated to 1        mL. The concentrate is dialyzed at 6° C. against 1 L of buffer        (5 mM sodium phosphate, 35 mM NaCl) in a dialysis tube for 16 h.        The dialysate is adjusted to an SDS content of 0.1% with a 2%        strength SDS solution in H₂O. The sample is removed by        centrifugation at 10000 g for 10 min and the supernatant is        removed.

8. Aβ(1-42) Fibrils

-   -   1 mg Aβ(1-42) (Bachem Inc. Cat. no.: H-1368) were solved in 500        μL aqueous 0.1% NH₄OH (Eppendorff tube) and the sample was        stirred for 1 min at room temperature. 100 μL of this freshly        prepared Aβ(1-42) solution were neutralized with 300 μL 20 mM        NaH₂PO₄; 140 mM NaCl, pH7.4. The pH was adjusted to pH 7.4 with        1% HCl. The sample was incubated for 24 h at 37° C. and        centrifuged (10 min at 10000 g). The supernatant was discarded        and the fibril pellet resuspended with 400 μL 20 mM NaH₂PO₄; 140        mM NaCl, pH 7.4 by vortexing for 1 min.

9. sAPPα

-   -   Supplied by Sigma (cat.no. S9564; 25 μg in 20 mM NaH₂PO₄; 140 mM        NaCl; pH 7.4). The sAPPα was diluted to 0.1 mg/mL (=1 pmol/μL)        with 20 mM NaH₂PO₄, 140 mM NaCl, pH 7.4, 0.2 mg/mL BSA.

Materials for Dot Blot: Aβ-Standards:

-   -   Serial dilution of Aβ antigens in 20 mM NaH₂PO₄, 140 mM NaCl, pH        7.4+0.2 mg/mL BSA        -   1) 100 pmol/μL        -   2) 10 pmol/μL        -   3) 1 pmol/μL        -   4) 0.1 pmol/μL        -   5) 0.01 pmol/μL

Nitrocellulose:

-   -   Trans-Blot Transfer medium, Pure Nitrocellulose Membrane (0.45        μm); BIO-RAD

Anti-Mouse-POD:

-   -   Cat. No: 715-035-150 (Jackson Immuno Research)

Detection Reagent:

-   -   BM Blue POD Substrate, precipitating (Roche)

Bovine Serum Albumin, (BSA):

-   -   Cat. No.: A-7888 (SIGMA)

Blocking Reagent:

-   -   5% low fat milk in TBS

Buffer Solutions:

  T B S  25  mM  Tris/HCl  buffer  pH  7.5 + 150  mM  NaCl  T T B S25  mM  Tris/HCl − buffer  pH  7.5 + 150  mM  NaCl + 0.05%  Tween  20  P B S + 0.2  mg/mL  B S A20  mM  NaH₂PO₄  buffer  pH  7.4 + 140  mM  NaCl + 0.2  mg/mL  B S A

Antibody Solution I:

-   -   0.2 μg/mL antibody diluted in 20 mL 1% low fat milk in TBS

Antibody Solution II:

-   -   1:5000 dilution    -   Anti-Mouse-POD in 1% low fat milk in TBS

Dot Blot Procedure:

-   -   1) 1 μL each of the different Aβ-standards (in their 5 serial        dilutions) were dotted onto the nitrocellulose membrane in a        distance of approximately 1 cm from each other.    -   2) The Aβ-standards dots were allowed to dry on the        nitrocellulose membrane on air for at least 10 min at room        temperature (RT) (=dot blot).    -   3) Blocking:        -   The dot blot was incubated with 30 mL 5% low fat milk in TBS            for 1.5 h at RT.    -   4) Washing:        -   The blocking solution was discarded and the dot blot            incubated under shaking with 20 mL TTBS for 10 min at RT.    -   5) Antibody Solution I:        -   The washing buffer was discarded and the dot blot incubated            with antibody solution I for 2 h at RT.    -   6) Washing:        -   The antibody solution I was discarded and the dot blot            incubated under shaking with 20 mL TTBS for 10 min at RT.            The washing solution was discarded and the dot blot            incubated under shaking with 20 mL TTBS for 10 min at RT.            The washing solution was discarded and the dot blot            incubated under shaking with 20 mL TBS for 10 min at RT.    -   7) Antibody Solution II:        -   The washing buffer was discarded and the dot blot incubated            with antibody solution II overnight at RT.    -   8) Washing:        -   The antibody solution II was discarded and the dot blot            incubated under shaking with 20 mL TTBS for 10 min at RT.            The washing solution was discarded and the dot blot            incubated under shaking with 20 mL TTBS for 10 min at RT.            The washing solution was discarded and the dot blot            incubated under shaking with 20 mL TBS for 10 min at RT.    -   9) Development:        -   The washing solution was discarded. The dot blot was            developed with 10 mL BM Blue POD Substrate for 10 min. The            development was stopped by intense washing of the dot blot            with H₂O. Quantitative evaluation was done using a            densitometric analysis (GS800 densitometer (BioRad) and            software package Quantity one, Version 4.5.0 (BioRad)) of            the dot-intensity. Only dots were evaluated that had a            relative density of greater than 20% of the relative density            of the last optically unambiguously identified dot of the            Aβ(20-42) globulomer. This threshold value was determined            for every dot-blot independently. The calculated value            indicates the relation between recognition of Aβ(1-42)            globulomer and the respective Aβ form for the antibody            given.

The monoclonal antibodies tested were obtained (except for 6E10) byactive immunization of mice with Aβ(12-42) globulomer (prepared asdescribed in Example I), followed by selection of the fused hybridomacells. The individual Aβ forms were applied in serial dilusions andincubated with the respective antibodies for immune reaction.

-   -   1. Aβ(1-42) monomer, 0.1% NH₄OH    -   2. Aβ(1-40) monomer, 0.1% NH₄OH    -   3. Aβ(1-42) monomer, 0.1% NaOH    -   4. Aβ(1-40) monomer, 0.1% NaOH    -   5. Aβ(1-42) globulomer    -   6. Aβ(12-42) globulomer    -   7. Aβ(20-42) globulomer    -   8. Aβ(1-42) fibril preparation    -   9. sAPPα (Sigma); (first dot: 1 pmol)

Results are shown in FIG. 1.

Based upon an analysis of the dot blot results, the anti-Aβ globulomermAbs 10F4 and 3C5 have a high affinity for Aβ-globulomer forms such asthe Aβ(1-42) globulomer, Aβ(12-42) globulomer and Aβ(20-42) globulomer).They discriminate other Aβ forms such as Aβ-monomers to a certain extentand do not significantly recognize Aβ(1-42) fibrils or sAPPα. Theantibodies 10F4 and 3C5 can therefore be coined ‘anti-Aβ globulomerantibodies’.

Example IV Detection of Aβ-Globulomer Epitopes in Alzheimer's DiseaseBrain by 10F4 AND 3C5 A: Extraction Procedure Reagent List:

-   -   3% SDS-buffer:        -   50 mM Tris/HCl, 150 mM NaCl, 0.5% Triton X100, 1 mM EGTA, 3%            SDS, 1% Na-desoxycholate, pH7.4    -   Complete Protease Inhibitor Cocktail:        -   dissolve 1 tablet complete inhibitor cocktail (Roche            Diagnostics GmbH; Cat. no.: 1697498) in 1 mL H₂O; freshly            prepared    -   PMSF-solution:        -   500 mM PMSF in methanol    -   3% SDS extraction-buffer:        -   add 1/100 complete inhibitor cocktail solution to the 3%            SDS-buffer        -   add 1/500 PMSF solution to the 3% SDS-buffer        -   prepare extraction buffer immediately before use at room            temperature    -   Antibodies:        -   mAb 10F4        -   mAb 3C5        -   mAb 6E10 (Signet; Cat. no.:9320)        -   mAb IgG2b (control antibody, generated against a synthetical            hapten, Dianova, clone NCG2B.01, Cat.No: DLN-05812)

Procedure:

0.2 g of −80° C. frozen post mortem human AD and aged match controlbrain tissue samples were added to 1.8 mL freshly prepared 3%SDS-extraction buffer at room temperature. The sample was immediatelyhomogenized on ice by a glass potter. The homogenized sample wastransferred to a reaction vial and centrifuged at 10000 g for 5 min. Thesupernatant (=3% SDS-brain extract) was collected carefully and storedin a reaction vial at −80° C. for further use.

B: Activation of Dynabeads with Monoclonal Mouse Antibodies

-   -   the stock-suspension of dynabeads (Dynabeads M-280 Sheep        anti-Mouse IgG, Invitrogen; Cat. no.: 112.02) was shaken        carefully to prevent foaming    -   1 mL was aseptically removed and transferred to a 1.5 mL        reaction vial    -   the dynabeads were washed 3 times 5 min with 1 mL        immunoprecipitation (IP)-wash buffer (IP-wash-buffer: PBS (20 mM        NaH₂PO₄, 140 mM NaCl, pH 7.4), 0.1% BSA). During the washing        procedure the supernatant was carefully removed while the        dynabeads were immobilized at the side of the reaction vial with        a magnetic separator stand (MSS)    -   the washed dynabeads were incubated with 40 μg Aβ-antibody in 1        mL PBS, 0.1% BSA    -   the activation was carried out by overnight incubation under        shaking at 4° C.    -   the activated dynabeads were washed 4 times 30 min (again using        the MSS) with 1 mL IP-wash buffer (PBS (20 mM NaH₂PO₄, 140 mM        NaCl, pH 7.4), 0.1% BSA)    -   the activated dynabeads were resuspended with 1 mL PBS, 0.1%        BSA, 0.02% Na-Azide; vortexed and centrifuged briefly    -   the antibody activated dynabeads were stored at 4° C. until        further use

C: Immunoprecipitation (IP)

-   -   25 μL 3% SDS-brain extract were diluted with 975 μL, 20 mM        NaH₂PO₄, 140 mM NaCl; 0.05% Tween 20, pH 7.5 (=1:40 dilution).    -   25 μL, of each antibody activated dynabeads of the following        list were incubated with 1 mL of the 1:40 diluted 3% SDS-brain        extract:        -   6E10-Dynabeads        -   3C5-Dynabeads        -   10F4-Dynabeads        -   IgG2b-Dynabeads    -   the immunoprecipitation was carried out by overnight incubation        (˜20 h) under shaking at 6° C.    -   the dynabeads were immobilized with the MPS    -   the supernatant was carefully removed and discarded    -   the dynabeads were washed as follows:        -   2 times 5 minutes with 500 μL 20 mM NaH₂PO₄, 140 mM NaCl, pH            7.5+0.1% BSA        -   1 time 3 minutes with 500 μL 2 mM NaH₂PO₄, 14 mM NaCl, pH            7.5        -   important: after the last removal of the washing buffer the            reaction vials were centrifuged, placed back in the MSS and            the remaining drops of fluid carefully removed        -   10 μL 50% CH₃CN, 0.5% TFA in H₂O were added to the reaction            vial and vortexed        -   the reaction vials were incubated 10 minutes at RT under            shaking        -   the dynabeads were immobilized with the MSS        -   the supernatant comprising the immunoprecipitated eluted Aβ            species was carefully withdrawn (=IP-eluate)

D: Surface-Enhanced Laser Desorption Ionization-Mass Spectrometry(SELDI-MS):

1 μL IP-eluate was spotted onto a H4 Protein Chip Array (Ciphergen;Cat.no. C573-0028).

-   -   the spots were allowed to dry on a warm incubator plate    -   CHCA-solution:        -   5 mg CHCA were dissolved in 150 μL acetonitrile+150 μL 1%            TFA=stock solution; stored at −20° C.        -   of the stock solution 10 μL were diluted with 20 μL            acetonitrile and 20 μL 1% TFA=working CHCA-solution        -   2 μL of the working CHCA-solution was applied onto the spots    -   the spots were allowed to dry on a warm incubator plate and        analysed by SELDI-MS (Surface-Enhanced Laser Desorption        Ionization-Mass Spectrometry)        -   conditions: laser intensity 200; sensitivity 6; mass range            800Da-10000Da; position 20-80; collect 5        -   analysis: the MZ area of the respective Aβ-mass peaks was            quantified

E. Western Blot Analysis of Imuunoprecipitated AD-Brain Extract:SDS-PAGE:

-   -   SDS-Sample buffer:        -   0.3 g SDS        -   4 mL 1 M Tris/HCl pH 6.8        -   8 mL glycerol        -   70 μL 1% bromphenolblue in ethanol        -   add H₂O to 50 mL    -   Running Buffer:        -   7.5 g Tris        -   36 g Glycine        -   2.5 g SDS        -   add H₂O to 2.5 L    -   SDS-PAGE Gel System:        -   18% Tris/Glycine Gel: (Invitrogen Inc., Cat. no.:            EC65055BOX)    -   5 μL IP-eluate were added to 13 μL sample buffer (300 μL        SDS-sample buffer+10 μL 1 M Tris-solution in H₂O+20 μL 85%        glycerol). The resulting 18 μL sample are loaded onto a 18%        Tris/Glycin Gel (Invitrogen Inc., Cat. no.: EC65055BOX). The        SDS-PAGE is conducted at a constant current of 20 mA.

Western Blot Procedure:

-   -   Subsequent to electrophoresis, the gel was blotted for 45        minutes at 75 mA onto a nitrocellulose membrane (7.5 cm×9 cm,        0.2 μm, BioRad) using a semi-dry blotting chamber (BioRad).    -   Blot-Buffer:        -   6 g Tris        -   28.1 g glycine        -   500 mL methanole        -   add H₂₀ to 2.5 L

Western Blot Immunostaining: Materials:

-   -   Anti-Aβ antibody 6E10 (Signet; Cat.No. 9320)    -   Anti-Mouse-POD (Jackson ImmunoResearch, Cat. no.: 715-035-150)    -   Detection reagent:        -   Super Signal West Pico Substrat (Pierce, Cat. no.: 34077)    -   Bovine Serum Albumin (BSA, Serva, Cat. no.: 11926)    -   low fat milk powder (Lasana)    -   Blocking reagent:        -   2% BSA in PBST    -   TBS:        -   25 mM Tris/HCl        -   150 mM NaCl Puffer, pH 7.5    -   TTBS:        -   25 mM Tris/HCl        -   150 mM NaCl Puffer        -   0.05% Tween 20, pH 7.5    -   PBS:        -   20 mM NaH₂PO₄ buffer        -   140 mM NaCl buffer, pH 7.5    -   PBST:        -   20 mM NaH₂PO₄ buffer        -   140 mM NaCl buffer        -   0.05% Tween 20, pH 7.5    -   Antibody solution I:        -   1 μg/mL 6E10=1:1000 in 20 mL 3% low fat milk in TBS    -   Antibody solution II:        -   1:10000 diluted anti-mouse-POD in 20 mL 3% low fat milk in            TBS

Procedure:

-   -   1) The Western blot was boiled for 10 minutes in PBS.    -   2) Blocking:        -   The Western blot was incubated for 16 h at 6° C. with 50 mL            blocking reagent.    -   3) Washing:        -   The blocking solution was discarded and the Western blot            washed with 50 mL TTBS for 10 minutes at room temperature.        -   The blocking solution was discarded and the Western blot            washed with 50 mL TBS for 10 minutes at room temperature.    -   4) Antibody solution I:        -   The washing solution was discarded and the Western blot            incubated with antibody solution I for 4 h at room            temperature.    -   5) Washing:        -   The blocking solution was discarded and the Western blot            washed with 50 mL TTBS for 10 minutes at room temperature.        -   The blocking solution was discarded and the Western blot            washed with 50 mL TTBS for 10 minutes at room temperature.        -   The blocking solution was discarded and the Western blot            washed with 50 mL TBS for 10 minutes at room temperature.    -   6) Antibody solution II:        -   The washing solution was discarded and the Western blot            incubated with antibody solution II for 1 h at room            temperature.    -   7) Washing:        -   The blocking solution was discarded and the Western blot            washed with 50 mL TTBS for 10 minutes at room temperature.        -   The blocking solution was discarded and the Western blot            washed with 50 mL TTBS for 10 minutes at room temperature.        -   The blocking solution was discarded and the Western blot            washed with 50 mL TBS for 10 minutes at room temperature.    -   8) Development and quantitative analysis:        -   The washing solution was discarded.        -   Two mL Super Signal West Pico Substrate Enhancer and 2 mL            Peroxide Solution were mixed.        -   The resulting 4 mL solution were added to the Western blot            and the blot was incubated for 5 minutes in the dark.        -   The blot was analyzed using a chemoluminescence imaging            system (VersaDoc, BioRad). Five pictures at were taken at            30, 97.5, 165, 232.5 and 300 seconds acquisition time.        -   The picture at which no saturation of the trace (intensity            x mm) of the Aβ-protein bands occurred was quantitatively            analyzed using the software package Quantity one, Version            4.5.0 (BioRad).            The results are shown in FIG. 2. The extraction procedure            with 3% (w/v) used herein is thought to extract soluble            forms of the total Aβ-peptide pool in the brain because the            buffer composition is not sufficient to solubilize            Aβ-peptide in the aggregated fibrillar form. The Aβ-peptide            that is bound in the Alzheimer's disease brain extract by            the monoclonal antibodies 3C5 and 10F4 is therefore soluble            Aβ-peptide. These soluble Aβ-species are thought to be the            Alzheimer's disease relevant species, as they correlate            better with the severity of the disease than fibrillar Aβ in            the form of Aβ-plaques found in AD brain (Kuo et al.            1996, J. Biol. Chem. 271, 4077-4081; Lue et al., 1999,            Am. J. Pathol. 155, 853-862). Therefore, the antibodies 10F4            and 3C5 target the disease relevant Aβ-species. Moreover, in            comparison to the pan-Aβ-antibody 6E10, the monoclonal            antibodies 3C5 and 10F4 bind only a to subfraction of the            total soluble Aβ-pool in the Alzheimer's disease brain            extract. The remaining Aβ-forms obviously do not possess the            Aβ-globulomer epitope recognized by 3C5 and 10F4. Due to the            fact that these Aβ-forms are not thought to be            neuropathogenic, it is advantegeous not to attack them by            the treatment antibody to reduce side effects and not to            reduce the effective concentration of antibodies circulating            in the CNS. Therefore, the dosing of the treatment antibody            can be reduced resulting in a better therapeutic index.

Example V Semi-Quantitative Analysis Visualized by SDS-Page of theDiscrimination of Anti-Aβ Globulomer Antibodies for Aβ(1-42) FibrilsAβ(1-42) Fibril Preparation:

1 mg of Aβ(1-42) (Bachem, Cat. No.: H-1368) was dissolved in 500 μL 0.1%NH₄OH in H₂O and agitated for 1 min at ambient temperature. The samplewas centrifuged for 5 min at 10000 g. The supernatant was collected.Aβ(1-42) concentration in the supernatant was determined according toBradford's method (BIO-RAD Inc. assay procedure).

100 μL of Aβ(1-42) in 0.1% NH₄OH were mixed with 300 μL of 20 mMNaH₂PO₄, 140 mM NaCl, pH 7.4 and adjusted to pH 7.4 with 2% HCl. Thesample was then incubated at 37° C. for 20 hours. Following which, thesample was centrifuged for 10 min at 10000 g. The supernatant wasdiscarded, and the residue was mixed with 400 L of 20 mM NaH₂PO₄, 140 mMNaCl, pH 7.4, resuspended by vigorous agitation (“vortexing”) for 1 minand centrifuged for 10 min at 10000 g. The supernatant was discarded,and the residue was mixed with 400 μL of 20 mM NaH₂PO₄, 140 mM NaCl, pH7.4, resuspended by vigorous agitation (“vortexing”) for 1 min andcentrifuged for 10 min at 10000 g once more. The supernatant wasdiscarded. The residue was resuspended in 380 L of 20 mM NaH₂PO₄, 140 mMNaCl, pH 7.4 and prompted by vigorous agitation (“vortexing”).

Binding of Anti-Aβ Antibodies to Aβ(1-42) Fibrils:

40 μL of Aβ(1-42) fibril preparation were diluted with 160 μL of 20 mMNaH₂PO₄, 140 mM NaCl, 0.05% Tween 20, pH 7.4 and agitated 5 min atambient temperature, and then the sample was centrifuged for 10 min at10000 g. The supernatant was discarded, and the residue was resuspendedin 95 L of 20 mM NaH₂PO₄, 140 mM NaCl, 0.05% Tween 20, pH 7.4.Resuspension was prompted by vigorous agitation (“vortexing”). Aliquotsof 10 μL of the fibril preparation were each mixed with:

-   -   a) 10 L 20 mM NaH₂PO₄, 140 mM NaCl, pH 7.4    -   b) 10 L 0.5 μg/μL of 3C5 in 20 mM NaH₂PO₄, 140 mM NaCl, pH 7.4    -   c) 10 L 0.5 μg/μL of 10F4 in 20 mM NaH₂PO₄, 140 mM NaCl, pH 7.4    -   d) 10 L 0.5 μg/μL of 6E10 (Signet Cat. Nr.: 9320) in 20 mM        NaH₂PO₄, 140 mM NaCl, pH 7.4        The samples were incubated at 37° C. for 20 hours, and then        centrifuged for 10 min at 10000 g. The supernatants were        collected and mixed with 20 μL of SDS-PAGE sample buffer. The        residues were mixed with 50 μL of 20 mM NaH₂PO₄, 140 mM NaCl,        0.025% Tween 20, pH 7.4 and resuspended by “vortexing”. Then,        the samples were centrifuged for 10 min at 10000 g. The        supernatants were discarded, and the residues were mixed with 20        μL 20 mM NaH₂PO₄, 140 mM NaCl, 0.025% Tween 20, pH 7.4, then        with 20 μL of SDS-PAGE sample buffer. The samples were applied        to a 4-20% Tris/glycine gel for electrophoresis.

Parameters for SDS-PAGE:

-   -   SDS sample buffer: 0.3 g SDS        -   4 mL 1M Tris/HCl pH 6.8        -   8 mL glycerine        -   1 mL 1% bromphenol blue in ethanol        -   Fill with H₂O ad 50 mL    -   4-20% Tris/Glycine Gel: (Invitrogen Cat.no.: EC6025BOX)    -   Electrophoresis buffer: 7.5 g Tris        -   36 g Glycine        -   2.5 g SDS        -   Fill with H₂O ad 2.5 L    -   The gel is run at a constant current of 20 mA.    -   Staining of the gels: Coomassie Blue R250

Results are shown in FIG. 3.

Semiquantitative Analysis of Different Anti-Aβ Antibodies and TheirDiscrimination of Aβ(1-42) Fibrils:

Positions of antibodies, Aβ(1-42) fibrils antibody heavy chain, antibodylight chain and Aβ(1-42) monomers are marked at the edge of the gel. Dueto their size, Aβ(1-42) fibrils cannot enter the SDS-PAGE gel and can beseen in the gel slot.

-   -   1. Marker    -   2. Aβ(1-42) fibril preparation; control    -   3. Aβ(1-42) fibril preparation; +mAb 6E10; 20 h 37° C.;        supernatant    -   4. Aβ(1-42) fibril preparation; +mAb 6E10; 20 h 37° C.; pellet    -   5. Aβ(1-42) fibril preparation; +mAb 3C5; 20 h 37° C.;        supernatant    -   6. Aβ(1-42) fibril preparation; +mAb 3C5; 20 h 37° C.; pellet    -   7. Aβ(1-42) fibril preparation; +mAb 10F4; 20 h 37° C.;        supernatant    -   8. Aβ(1-42) fibril preparation; +mAb 10F4; 20 h 37° C.; pellet        The relative binding to fibril type Aβ was evaluated from        SDS-PAGE analysis by measuring the Optical Density (OD) values        from the Heavy Chain of the antibodies in the fibril bound        (pellet-fraction) and the supernatant fractions after        centrifugation. Antibodies that have bound to the Aβ fibrils        should be co-pelleted with the Aβ-fibrils and therefore are        found in the pellet fraction whereas non-Aβ-fibril bound (free)        antibodies are found in the supernatant. The percentage of        antibody bound to Aβ-fibrils was calculated according to the        following formula:

Percent antibody bound toAβ-fibrils=OD_(fibril fraction)×100%/(OD_(fibril fraction)+OD_(supernatant fraction)).

Results are shown in FIG. 3. In contrast to the commercially availableantibody 6E10 (Signet Cat. no.: 9320) which recognizes and binds to alinear Aβ-epitope between AA1-17, the Aβ globulomer antibodies 3C5 and10F4 bind to Aβ(1-42)-fibrils with a lower affinity in a co-pelletingexperiment. This is evidenced by the fact that the 3C5 and 10F4antibodies, after an incubation with Aβ(1-42) fibrils, remain mainlyafter a pelleting step in the supernatant and are not co-pelleted due tobeing bound to the Aβ(1-42) fibrils.

In the Alzheimer's disease brain, the Aβ fibrils are a major componentof the total Aβ peptide pool. By attacking these fibrils by antiAβ-antibodies, the risk of negative side effects is elevated due to aliberation of high amounts of Aβ which subsequently may increase therisk of microhaemorrhages. An increased risk for microhemorrhages wasobserved in an active immunization approach with fibrillar aggregates ofthe Aβ peptide (Bennett and Holtzman, 2005, Neurology, 64, 10-12;Orgogozo J, Neurology, 2003, 61, 46-54; Schenk et al., 2004, Curr OpinImmunol, 16, 599-606).

Example VI In Situ Analysis of the Specific Reaction of Antibodies 10F4and 3C5 to Fibrillar Abeta Peptide in the Form of Amyloid Plaques andAmyloid in Meningeal Vessels in Old App Transgenic Mice And Alzheimer'SDisease Patients

Antibodies 10F4 and 3C5 show reduced staining to fibrillar Aβ peptidedeposits suggesting that their therapeutic effect is mediated by bindingto soluble globulomeric forms rather than fibrillar deposited forms ofAβ peptide. Since antibody binding to fibrillar Aβ peptide can lead tofast dissolution of aggregates and a subsequent increase of soluble Aβconcentration, which in turn is thought to be neurotoxic and could leadto microhemorrhages, an antibody therapy that effects the solubleglobulomer rather than the monomer is preferred.

Methods:

For these experiments, several brain material samples were used:cortical tissue from 2 AD patients (RZ16 and RZ 55) and cortical tissuefrom 19 month old Tg2576 mice (APPSWE #001349, Taconic, Hudson, N.Y.,USA) or 12 month old APP/L mice (ReMYND, Leuven, Belgium).

The mice overexpress human APP with a familial Alzheimer's diseasemutation and form β-amyloid deposits in the brain parenchyma at about 11months of age and β-amyloid deposits in larger cerebral vessels at about18 months of age. The animals were deeply anaesthetized andtranscardially perfused with 0.1 M phosphate-buffered saline (PBS) toflush the blood. Then, the brain was removed from the cranium anddivided longitudinally. One hemisphere of the brain was shock-frozen andthe other fixated by immersion into 4% paraformaldehyde. Theimmersion-fixated hemisphere was cryoprotected by soaking in 30% sucrosein PBS and mounted on a freezing microtome. The entire forebrain was cutinto 40 μm transverse sections which were collected in PBS and used forthe subsequent staining procedure.

The neocortex samples from Alzheimer's disease patients were obtainedfrom Brain-Net, Munich, Germany as frozen tissue, immersion-fixated in4% paraformaldehyde during thawing, and subsequently treated like themouse tissue.

Individual Sections were Stained with Congo Red Using the FollowingProtocol:

Material:

-   -   Amyloid dye Congo Red kit (Sigma-Aldrich; HT-60), consisting of        alcoholic NaCl solution, NaOH solution and Congo Red solution    -   staining cuvettes    -   microscope slides SuperfrostPlus and coverslips    -   Ethanol, Xylol, embedding medium

Reagents:

-   -   NaOH diluted 1:100 with NaCl solution yields alkaline saline    -   alkaline saline diluted 1:100 with Congo Red solution yields        alkaline Congo Red solution (prepare no more than 15 min before        use, filtrate)    -   mount sections on slide and allow them to dry    -   incubate slide in staining cuvette, first for 30-40 minutes in        alkaline saline, then for 30-40 minutes in alkaline Congo Red        solution    -   rinse three times with fresh ethanol and embed over xylol

Staining was first photographed using a Zeiss Axioplan microscope(Zeiss, Jena, Germany) and evaluated qualitatively. Red colour indicatedamyloid deposits both in the form of plaques and in larger meningealvessels. Later on, evaluation of antibody staining focused on thesestructures.

Staining was performed by incubating the sections with a solutioncontaining 0.07-0.7 μg/ml of the respective antibody in accordance withthe following protocol:

Materials:

-   -   TBST washing solution (Tris Buffered Saline with Tween 20; 10×        concentrate; DakoCytomation 53306, DAKO, Hamburg, Germany) 1:10        in Aqua bidest.)    -   0.3% H₂O₂ in methanol    -   donkey serum (Serotec, Düsseldorf, Germany), 5% in TBST, as        blocking serum    -   monoclonal mouse-anti-globulomer antibodies diluted at given        concentrations in TBST    -   secondary antibody: biotinylated donkey-anti-mouse antibody        (Jackson Immuno/Dianova, Hamburg, Germany; 715-065-150; diluted        1:500 in TBST)    -   StreptABComplex (DakoCytomation K 0377, DAKO, Hamburg, Germany)    -   Peroxidase Substrate Kit diaminobenzidine (=DAB; SK-4100; Vector        Laboratories, Burlingame, Calif., USA)    -   SuperFrost Plus microscope slides and coverslips    -   xylol free embedding medium (Medite, Burgdorf, Germany; X-tra        Kitt)

Procedure:

-   -   transfer floating sections into ice-cold 0.3% H₂O₂ and incubate        for 30 min    -   wash for 5 min in TBST buffer    -   incubate with donkey serum/TBST for 20 minutes    -   incubate with primary antibody for 24 hours at room temperature    -   wash in TBST buffer for 5 minutes    -   incubate with blocking serum for 20 minutes    -   wash in TBST buffer for 5 minutes    -   incubate with secondary antibody for 60 minutes at ambient        temperature    -   wash in TBST buffer for 5 minutes    -   incubate with StreptABComplex for 60 minutes at ambient        temperature    -   wash in TBST buffer for 5 minutes    -   incubate with DAB for 20 minutes    -   mount the section on slides, air-dry slides, dehydrate slides        with alcohol and embed slides

Besides visual inspection of sections under the microscope, amyloidstaining was additionally quantified by optically excising 10 randomlyselected plaques from the histological images using the ImagePro 5.0image analysis system and determining their average greyscale value.Optical density values (were calculated from the greyscale values bysubtracting the mean background density of the stained material from thedensity of amyloid plaques (0%—no plaque staining above surroundingbackground, 100%—no transmission/maximal staining). The differencesbetween antibodies 6E10/4G8 and 6G1, 10F4 and 3C5, respectively, weretested for statistical significance with ANOVA.

Results:

All antibody stained material described in the following proved to becongophilic amyloid deposits (FIG. 4( a)). The globulomer-preferringantibodies 10F4 and 3C5 stained parenchymal and meningeal congophilicdeposits of Aβ peptide at the same concentration of 0.7 μg/mLsignificantly less than the antibodies 6G1 and 6E10 (FIG. 4( b,c,h)).Quantitative analysis of parenchymal amyloid plaque staining revealedbinding of all antibodies to plaques (statistically significant densityabove control), but binding of antibody 10F4 and 3C5 was significantlylower than binding of the reference antibody 6E10 (raised to N-terminalsequence of Aβ) and equal or lower than reference antibody 4G8 (raisedto N-terminal sequence of Aβ) (FIG. 4( d-g)).

Antibodies 10F4 and 3C5 bind less to amyloid deposits than antibodieswhich recognize Aβ monomer or part of the Aβ sequence. Treatment withantibodies binding to fibrillar Aβ peptide can lead to fast dissolutionof amyloid plaques in brain tissue and a subsequent increase of solubleAβ concentration, which in turn is thought to be neurotoxic and couldlead to microhemorrhages, and/or a fast dissolution of vascular amyloid,which also could lead to microhemorrhages. Therefore, an antibodytherapy that effects the soluble globulomer rather than the monomer ispreferred.

Example VII Endogenous Aβ(1-42) and Aβ(1-40) Levels in CSF of ADpatients After Immunoprecipitation with Anti-Aβ Globulomer Antibodies10F4 and 3C5

Immunoprecipitation (IP) of Aβ-species from AD-brain CSF with DynabeadsM-280 Sheep anti-Mouse IgG

The following mAbs were immobilized to Dynabeads M-280 Sheep anti-MouseIgG:

-   -   mAb 6E10 (Signet Inc.; Cat. no.: 9320)    -   mAb 3C5    -   mAb 10F4    -   mAb 8F5

Dynabeads M-280 Sheep Anti-Mouse IgG:

Sheep anti-Mouse IgG (Invitrogen Inc., Cat. no.: 112.02) is covalentlybound to magnetic beads (Dynabeads).

Activation of Dynabeads with Monoclonal Mouse Antibodies

-   -   the stock-suspension of dynabeads (Dynabeads M-280 Sheep        anti-Mouse IgG, Invitrogen; Prod. No. 112.02) was shaken        carefully to prevent foaming    -   1 mL was aseptically removed and transferred to a 1.5 mL        reaction vial    -   the dynabeads were washed 3 times 5 min with 1 mL        immunoprecipitation (IP)-wash buffer (IP-wash-buffer: PBS (20 mM        NaH₂PO₄, 140 mM NaCl, pH 7.4), 0.1% (w/v) BSA). During the        washing procedure the supernatant was carefully removed while        the dynabeads were immobilized at the side of the reaction vial        with a magnetic separator stand (MSS)    -   the washed dynabeads were incubated with 40 μg Aβ-antibody in 1        mL PBS, 0.1% (w/v) BSA    -   the activation was carried out by overnight incubation under        shaking at 4° C.    -   the activated dynabeads were washed 4 times 30 min (again using        the MSS) with 1 mL IP-wash buffer (PBS (20 mM NaH₂PO₄, 140 mM        NaCl, pH 7.4), 0.1% (w/v) BSA)    -   the activated dynabeads were resuspended with 1 mL PBS, 0.1%        (w/v) BSA, 0.02% (w/v) Na-Azide; vortexed and centrifuged        briefly    -   the antibody activated dynabeads were stored at 4° C. until        further use

CSF Sample Preparation:

400 μL CSF from an Alzheimer's disease patient were added to 4 μLComplete Protease Inhibitor Cocktail (Roche Inc. Cat. no.: 1697498, 1tablet dissolved in 1 mL water) and 0.8 μL 500 mM PMSF dissolved inmethanol. After 10 min 1.6 mL 20 mM NaH₂PO₄,140 mM NaCl, 0.05% Tween 20,pH 7.4 (PBST) was added.

Immunoprecipitation of Aβ Species from Human AD-CSF:

250 μL aliquot of the prepared CSF sample were added to 25 μLanti-Aβ-Dynabeads suspension

-   -   Immunoprecipitation occurred under stirring at 6° C. for 16        hours. Subsequent washing of the beads was performed 3 times 5        min. with 1 mL PBS/0.1% (w/v) BSA and finally once 3 min. with 1        mL 10 mM Tris/HCL pH 7.5 buffer. During the washing procedure        the supernatant was carefully removed while the dynabeads were        immobilized at the side of the reaction vial with a magnetic        separator stand (MSS)

The residual supernatant was thoroughly removed after the final washingstep.

The Aβ peptides and the corresponding antibody were removed from theDynabeads by adding 25 μL sample buffer without β-Mercaptoethanol (0.36M Bistris, 0.16 M Bicine, 1% SDS (w/v), 15% (w/v) sucrose, 0.004% (w/v)Bromphenolblue) to the Eppendorff tube and heating for 5 min at 95° C.in a heating block. After cooling to room temperature the dynabeads wereimmobilized at the side of the reaction vial with a magnetic separatorstand (MSS) and the supernatant were transferred to another Eppendorfftube (IP eluate).

Analysis of Aβ Immunoprecipitates by Urea-PAGE Followed by Western BlotProcedure:

The quantification of Aβ1-40 and Aβ1-42 species was performed by a 8 MUrea Poly-Acrylamide-Gel-Electrophoresis system and subsequent WesternBlot analysis according to the procedure first described by H. W. Klafkiet al., Analytical Biochemistry 237., 24-29 (1996) and later also usedby J. Wiltfang et al., J. of Neurochemistry 81, 481-496, 2002. Therewere only two minor changes made in the experimental procedure:

-   -   1) SDS concentration in the stacking gel was adjusted to 0.25%        (w/v) instead of 0.1% (w/v).    -   2) For the Western blot the antibody 1E8 (Senetek Drug Delivery        Technologies Inc. St. Louis, Mo., USA) was replaced by        Anti-Human Amyloid β (N) (82E1) Mouse IgG mAb (IBL, Cat.no.:        10323)

15 μL IP eluate aliquots of the immunoprecipitated samples were loadedonto the 8 M Urea PAGE. Electrophoresis was performed at 100 V (15 min)and continued at 60 V. The electrophoresis was stoppep when the runningfront of the blue sample loading dye was still 0.5 cm away from the endof the gel.

Western Blot Procedure:

Western blot analysis was performed in a Semi Dry Blotting chamber(BioRad Inc., 45 min at 75 mA) onto 7.5 cm×9 cm Nitrocellulose 0.45 μm(BioRad Inc.)

Blotting buffer: 6 g Tris; 28.1 g Glycin; 500m L Methanol; adjust to 2.5l with water.

The Nitrocellulose blot was boiled for 10 min in PBS at 100° C. The blotwas saturated by treatment with 50 mL 5% (w/v) BSA in PBST for 1 hour atRT. After removal of the fluid phase the following washing step wereperformed twice with: 50 mL TTBS (25 mM Tris/HCl; 150 mM NaCl Puffer;0.05% Tween 20; pH 7.5) for 10 min at RT and subsequently with 50 mL TBS(25 mM Tris/HCl; 150 mM NaCl buffer; pH 7.5) for 10 min at RT.

For further development the final washing buffer was discarded from theblot and 15 mL antibody I solution (0.2 μg/mL 82E1=1:500 in 3% (w/v)skimmed milk powder (Lasana Inc.), in 15 mL TBS) were added for 20 hoursat 6° C. Removal of buffer was followed by the three wash steps asdescribed above. The blot was incubated with Antibody solution II(1:10000 dilution of anti-Mouse-POD in 15 mL 3% (w/v) skimmed milkpowder in 15 mL TBS) for 1 hour at RT. Removal of buffer was followed bythe three wash steps as described above.

After removal of the last washing buffer 2 mL Super Signal West FemtoMaximum Sensitivity Substrat Enhancer and 2 mL Peroxide Solution wasmixed. The freshly prepared solution was poured onto the blot which waspreincubated in the dark for 5 min. Chemoluminescence was recorded usinga VersaDoc Imaging system (BioRad).

Imaging Parameters:

exposure time 180 sec.Picture records after 30 sec., 60 sec., 120 sec. and 180 sec.

The results were obtained from the picture with 180 sec. exposure time.

The anti-globulomer antibodies 10F4 and 3C5 of the present inventionhave a lower affinity for Aβ(1-42) peptide and Aβ(1-40) peptide in theCSF of an Alzheimer's disease patient, in comparison to the commerciallyavailable antibody 6E10 (which is, in the literature, regarded torecognize all Aβ-forms regardless of their conformation). CSF Aβ-peptideforms undergo a high turnover rate (Bateman et al., Nature Medicine,2006, 12(7):856-61) and are therefore unlikely the disease relavantspecies. Therefore, the CSF Aβ-forms should not be targeted in a passiveimmunization treatment strategy of Alzheimer's disease in order toreduce the risk of undesired side effects. It is noted that, in anearlier study (Barghorn et al., J. Neurochem. 2005; 95(3):834-847), theanti Aβ-globulomer antibody 8F5 did not recognize and bind to Aβ-peptidein the CSF of an Alzheimer's disease patient. This earlier study wasperformed using a sandwich ELISA method. In contrast, when using theimmunoprecipitation and Urea PAGE method described above, the sameantibody 8F5 does recognize Aβ-peptide in the CSF of an Alzheimer'sdisease patient (see FIG. 5). Therefore, the sandwich ELISA methodproduced false negative results; hence, for the detection of Aβ-peptidesin CSF, the immunoprecipitation and Urea PAGE methods described hereinshould be used.

What is claimed is:
 1. An isolated antibody having a higher affinity toAβ(1-42) globulomer than to at least one amyloid beta protein selectedfrom the group consisting of Aβ(1-42) peptide present in cerebrospinalfluid (CSF) and b) Aβ(1-40) peptide present in CSF.
 2. An isolatedantibody having a binding affinity to Aβ(1-42) globulomer which isgreater than the binding affinity to at least one amyloid beta proteinselected from the group consisting of a) Aβ(1-42) monomer, b) Aβ(1-40)monomer, c) Aβ(1-42) fibril and d) soluble amyloid precursorprotein-alpha (sAPPα).
 3. The isolated antibody of claim 2 wherein saidantibody binds with less affinity to amyloid beta protein present innon-CSF than to amyloid beta protein present in CSF.
 4. The isolatedantibody of claim 1 or claim 2, wherein the antibody is a monoclonalantibody.
 5. The isolated antibody of claim 4, wherein the antibody is arecombinant antibody.
 6. The isolated antibody of claim 1 or claim 2,wherein the antibody is human or humanized.
 7. The isolated antibody ofclaim 1 or claim 2, wherein said antibody binds to at least one epitopeas a monoclonal antibody selected from the group consisting of themonoclonal antibody 10F4 obtainable from a hybridoma designated byAmerican Type Culture Collection deposit number PTA-7808 and themonoclonal antibody 3C5 obtainable from a hybridoma designated byAmerican Type Culture Collection deposit number PTA-7406.
 8. An isolatedantibody comprising SEQ ID NO:5.
 9. An isolated antibody comprising SEQID NO:6.
 10. The isolated antibody of claim 9 further comprising SEQ IDNO:5.
 11. An isolated antibody comprising SEQ ID NO:7.
 12. An isolatedantibody comprising SEQ ID NO:8.
 13. The isolated antibody of claim 12further comprising SEQ ID NO:7.
 14. The isolated antibody of claim 1 orclaim 2, wherein the antibody comprises at least one amino acid sequenceselected from the group consisting of: a) the amino acid sequence of theheavy chain CDR3 and the amino acid sequence of the light chain CDR3 ofa monoclonal antibody (10F4) obtainable from a hybridoma designated byAmerican Type Culture Collection deposit number PTA-7808 and b) theamino acid sequence of the heavy chain CDR3 and the amino acid sequenceof the light chain CDR3 of a monoclonal antibody (3C5) obtainable from ahybridoma designated by American Type Culture Collection deposit numberPTA-7406.
 15. The isolated antibody of claim 1 or claim 2, wherein theantibody comprises at least one amino acid sequence selected from thegroup consisting of: a) the amino acid sequence of the heavy chain CDR2and the amino acid sequence of the light chain CDR2 of a monoclonalantibody (10F4) obtainable from a hybridoma designated by American TypeCulture Collection deposit number PTA-7808 and b) the amino acidsequence of the heavy chain CDR2 and the amino acid sequence of thelight chain CDR2 of a monoclonal antibody (3C5) obtainable from ahybridoma designated by American Type Culture Collection deposit numberPTA-7406.
 16. The isolated antibody of claim 1 or claim 2, wherein theantibody comprises at least one amino acid sequence selected from thegroup consisting of: a) the amino acid sequence of the heavy chain CDR1and the amino acid sequence of the light chain CDR1 of a monoclonalantibody (10F4) obtainable from a hybridoma designated by American TypeCulture Collection deposit number PTA-7808 and b) the amino acidsequence of the heavy chain CDR1 and the amino acid sequence of thelight chain CDR1 of a monoclonal antibody (3C5) obtainable from ahybridoma designated by American Type Culture Collection deposit numberPTA-7406.
 17. An isolated antibody comprising at least one CDR selectedfrom the group consisting of amino acid sequence: a) SHYAWN; b)YIDYSGSTRYLPSLKS; c) GSGYFYGMDY; d) HASQNINVWLS; e) KASNLHT; f)QQGQSYPYT; g) NYLIE; h) VINPGSGDTNYNENFKG; i) GVITTGFDY; j) RASGNIHNYLA;k) NAKTLAD and l) QHFWSSPRT.
 18. A hybridoma designated by American TypeCulture Collection deposit number PTA-7808.
 19. A monoclonal antibody(10F4) obtainable from a hybridoma designated by American Type CultureCollection deposit number PTA-7808.
 20. A hybridoma designated byAmerican Type Culture
 21. A monoclonal antibody (3C5) obtainable from ahybridoma designated by American Type Culture Collection deposit numberPTA-7406.
 22. An isolated nucleic acid molecule encoding the antibody ofclaim 1 or claim
 2. 23. The isolated nucleic acid molecule of claim 22wherein the nucleotide sequence of said molecule comprises at least onesequence selected from the group consisting of SEQ ID NO:1, SEQ ID NO:2,SEQ ID NO:3 and SEQ ID NO:4.
 24. A vector comprising said isolatednucleic acid molecule of claim
 23. 25. A host cell comprising saidvector of claim
 24. 26. A method of producing an antibody, comprisingculturing said host cell of claim 25 in a culture medium for a time andunder conditions suitable for production of said antibody.
 27. Anisolated antibody produced by said method of claim
 26. 28. A compositioncomprising said antibody of claim 1, said antibody of claim 2 or acombination thereof.
 29. The composition of claim 28, wherein thecomposition is a pharmaceutical composition and further comprises apharmaceutical acceptable carrier.
 30. A monoclonal antibody comprisingan amino acid sequence encoded by at least one nucleotide sequenceselected from the group consisting of SEQ ID NO:1, SEQ ID NO:2, SEQ IDNO:3 and SEQ ID NO:4.
 31. The isolated antibody of claim 30 wherein saidantibody is selected from the group consisting of a monoclonal antibodyproduced by a hybridoma designated by American Type Culture Collectiondeposit number PTA-7406 and a monoclonal antibody produced by ahybridoma designated by American Type Culture Collection deposit numberPTA-7808.
 32. The isolated antibody of claim 30 wherein said antibodycomprises at least one amino acid sequence selected from the groupconsisting of SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7 and SEQ ID NO:8. 33.A method for treating or preventing an amyloidosis in a patient in needof said treatment or prevention comprising administering said antibodyof claim 1 or claim 2 to said patient in an amount sufficient to effectsaid treatment or prevention.
 34. The method of claim 33 wherein saidamyloidosis is Alzheimer's disease or the amyloidosis of Down'ssyndrome.
 35. An isolated antibody which binds to at least one epitopeof amyloid beta protein in the brain of a patient having amyloidosis.36. The isolated antibody of claim 35, wherein said antibody is producedby a hybridoma having an ATCC deposit number selected from the groupconsisting of PTA-7406 and PTA-7808.
 37. A method of diagnosingAlzheimer's Disease in a patient suspected of having this diseasecomprising the steps of: a. isolating a biological sample from saidpatient b. contacting said biological sample with said isolated antibodyof claim 1 or claim 2 for a time and under conditions sufficient forformation of antigen/antibody complexes; and c. detecting presence ofsaid antigen/antibody complexes in said sample, presence of saidcomplexes indicating a diagnosis of Alzheimer's Disease in said patient.38. The method of claim 37 wherein said antigen is a globulomer.
 39. Amethod of diagnosing Alzheimer's Disease in a patient suspected ofhaving this disease comprising the steps of: a. isolating a biologicalsample from said patient; b. contacting said biological sample with anantigen for a time and under conditions sufficient for the formation ofantibody/antigen complexes; c. adding a conjugate to the resultingantibody/antigen complexes for a time and under conditions sufficient toallow said conjugate to bind to the bound antibody, wherein saidconjugate comprises said isolated antibody of claim 1 or claim 2,attached to a signal generating compound capable of generating adetectable signal; and d. detecting the presence of an antibody whichmay be present in said biological sample, by detecting a signalgenerated by said signal generating compound, said signal indicating adiagnosis of Alzheimer's Disease in said patient.
 40. The method ofclaim 28 wherein said antigen is a globulomer.
 41. A method ofdiagnosing Alzheimer's Disease in a patient suspected of havingAlzheimer's Disease comprising the steps of: a. isolating a biologicalsample from said patient; b. contacting said biological sample withanti-antibody, wherein said anti-antibody is specific for said antibodyof claim 1 or claim 2, for a time and under conditions sufficient toallow for formation of anti-antibody/antibody complexes, said complexescontaining antibody present in said biological sample; c. adding aconjugate to resulting anti-antibody/antibody complexes for a time andunder conditions sufficient to allow said conjugate to bind to boundantibody, wherein said conjugate comprises an antigen, which binds to asignal generating compound capable of generating a detectable signal;and d. detecting a signal generated by said signa generating compound,said signal indicating a diagnosis of Alzheimer's Disease in saidpatient.
 42. A vaccine comprising: a) said isolated antibody of claim 1,said isolated antibody of claim 2 or a combination thereof and b) apharmaceutically acceptable adjuvant.
 43. A method of identifyingcompounds suitable for active immunization of a patient predicted todevelop Alzheimer's Disease comprising the steps of: a) exposing one ormore compounds of interest to said isolated antibody of claim 1 or claim2, for a time and under conditions sufficient for said one or morecompounds to bind to said isolated antibody of claim 1 or claim 2; andb) identifying those compounds which bind to said isolated antibody ofclaim 1 or claim 2, said identified compounds to be used in activeimmunization in a patient predicated to develop Alzheimer's Disease. 44.A kit comprising: a) said isolated antibody of claim 1 or claim 2 and b)a conjugate comprising an antibody attached to a signal-generatingcompound, wherein said antibody of said conjugate is different from saidisolated antibody.
 45. A kit comprising: a) an anti-antibody to saidisolated antibody of claim 1 or claim 2 and b) a conjugate comprising anantigen attached to a signal-generating compound.
 46. The kit of claim46 wherein said antigen is a globulomer.